THE    PRINCIPLES 


OF 


BACTERIOLOGY: 


A  PRACTICAL  MANUAL  FOR  STUDENTS  AND 
PHYSICIANS. 


BY 

A.   C.   ABBOTT,   M.D., 

FIRST  ASSISTANT,  LABORATORY  OP  HYGIENE,  UNIVERSITY  OP  PENNSYLVANIA, 
PHILADELPHIA. 


THIRD  EDITION,  ENLARGED  AND   THOROUGHLY 
REVISED. 

With  98  Illustrations,  of  which   17  are  colored. 


PHILADELPHIA: 

LEA    BROTHERS    &    CO 
1895. 


BIOLOGY 

R 

G 


Entered  according  to  the  Act  of  Congress,  in  the  year  1895,  by 

LEA  BROTHERS  &  CO., 
In  the  Office  of  the  Librarian  of  Congress.    All  rights  reserved. 


Main 


ib.-^ 


PHILADELPHIA  : 
DOBNAN,    PRINTER. 


Of    THE 

UNIVERSITY 

OF 

Llf 


PREFACE  TO  THE  THIRD  EDITION, 


THE  call  for  a  third  edition  of  this  book,  coming  so 
soon  after  the  appearance  of  the  second  edition,  is  a 
source  of  no  small  degree  of  gratification  to  the  author. 

During  the  interval  of  about  twelve  months  that  has 
elapsed  since  the  appearance  of  the  second  edition  ad- 
vances have  been  made  along  the  manifold  lines  of 
bacteriological  research,  but  they  have  not  been  of  a 
nature  to  cause  any  radical  modification  of  the  funda- 
mental principles  involved.  Where  such  advances  have 
come  within  the  scope  of  a  book  of  this  character  an 
effort  has  been  made  to  present  them,  but  the  original 
aim  and  object  of  the  book  have  been  maintained. 

The  author  wishes  to  acknowledge  his  indebtedness 
to  the  writers  of  the  many  flattering  reviews  that  "  The 
Principles  of  Bacteriology"  has  received,  and  hopes 
that  in  future,  as  in  the  past,  readers  will  continue  to 
favor  him  with  criticism  and  suggestions  that  may  serve 
to  further  enhance  the  value  of  the  work. 

A.  C.  A. 

PHILADELPHIA,  NOVEMBER,  1895. 


208314 


PREFACE  TO  THE  SECOND  EDITION. 


THE  cordial  reception  with  which  this  book  has  met, 
and  the  demand  for  a  second  edition,  afford  the  author 
no  small  degree  of  gratification.  In  revising  The  Prin- 
ciples of  Bacteriology  advantage  has  been  taken  of  the 
valuable  suggestions  kindly  offered  by  the  reviewers  of 
the  first  edition,  for  which  the  writer  here  acknowledges 
his  indebtedness. 

The  section  of  the  work  devoted  to  descriptive  bac- 
teriology has  been  somewhat  extended,  but  no  effort  has 
been  made  to  cover  the  entire  field,  only  those  species 
being  introduced  that  are  comparatively  common  or  of 
importance  in  enabling  the  student  to  acquire  a  funda- 
mental working  knowledge  capable  of  wider  application. 
Wherever  practicable,  these  descriptions  have  been  sup- 
plemented by  illustrations,  for  the  majority  of  which 
the  author  is  responsible.  The  introduction  of  colored 
figures  in  the  text  is  a  new  feature  in  this  edition,  and 
one  which  should  increase  its  usefulness.  A  sketch  of 
the  evolution  of  our  knowledge  upon  immunity  and 
infection  has  been  introduced,  and  an  outline  of  appa- 
ratus necessary  for  a  beginner's  laboratory  has  been 
appended. 


vi  PREFACE  10  THE  SECOND  EDITION. 

The  original  purpose  of  this  book  has  been  main- 
tained, and  it  is  hoped  that  the  second  edition,  contain- 
ing double  the  letterpress  and  treble  the  number  of 
illustrations  fouud  in  its  predecessor,  will  in  some  cor- 
responding measure  improve  upon  the  service  which 
the  work  has  apparently  rendered  to  students  and 
physicians. 

A.  C   A. 

PHILADELPHIA,  July,  1894. 


PREFACE  TO  THE  FIRST  EDITION. 


IN  preparing  this  book  the  author  has  kept  in  mind 
the  needs  of  the  student  and  practitioner  of  medicine, 
for  whom  the  importance  of  an  acquaintance  with 
practical  bacteriology  cannot  be  overestimated. 

It  is  to  advances  made  through  bacteriological  re- 
search that  we  are  indebted  for  much  of  our  knowledge 
of  the  conditions  underlying  infection,  and  for  the 
elucidation  of  many  hitherto  obscure  problems  con- 
cerning the  etiology,  the  modes  of  transmission,  and 
the  means  of  prevention  of  infectious  maladies. 

Only  within  a  comparatively  short  time  have  students 
and  physicians  been  enabled  to  obtain  the  systematic 
instruction  in  this  science  that  is  of  value  in  aiding 
them  in  their  efforts  to  check  disease.  The  rapid  in- 
crease in  the  number  who  are  availing  themselves  of 
these  opportunities  speaks  directly  for  the  practical 
value  of  the  science. 

As  the  majority  of  those  undertaking  the  study  of 
bacteriology  do  so  with  the  view  of  utilizing  it  in 
medical  practice,  and  as  many  of  these  can  devote  to  it 
but  a  portion  of  their  time,  it  is  desirable  that  the 
subject-matter  be  presented  in  as  direct  a  manner  as 
possible. 


viii  PREFACE  TO  THE  FIRST  EDITION. 

Presuming  the  reader  to  be  unfamiliar  with  the  sub- 
ject, the  author  has  restricted  himself  to  those  funda- 
mental features  that  are  essential  to  its  understanding. 
The  object  has  been  to  present  the  important  ideas  and 
methods  as  concisely  as  is  compatible  with  clearness, 
and  at  the  same  time  to  accentuate  throughout  the 
underlying  principles  which  govern  the  work. 

With  the  view  of  inducing  independent  thought  on 
the  part  of  the  student,  and  of  diminishing  the  fre- 
quency of  that  oft-heard  query,  "What  shall  I  do 
next?"  experiments  have  been  suggested  wherever  it 
is  possible.  These  have  been  arranged  to  illustrate  the 
salient  points  of  the  work  and  to  attract  attention  to 
the  minute  details,  upon  the  observation  of  which  so 
much  in  bacteriology  depends. 

A.  C.  A. 
PHILADELPHIA,  December,  1891. 


CONTENTS. 


INTRODUCTION. 


The  overthrow  of  the  doctrine  of  spontaneous  generation—"  Omne 
vivum  ex  vivo  " — Earlier  bacteriological  studies — The  birth  of  mod- 
ern bacteriology  13-2G 


CHAPTEK   I. 

Definition  of  bacteria— Their  place  in  nature— Difference  between 
parasites  and  saprophytes— Nutrition  of  bacteria— Products  of  bac- 
teria—Their relation  to  oxygen— Influence  of  temperature  upon 
their  growth 27-35 

CHAPTEE    II. 

Morphology  of  bacteria— Grouping— Mode  of  multiplication— 
Spore-formation-Motility  .  36-46 


CHAPTER    III. 

Principles  of  sterilization  by  heat— Methods  employed  —  Discon- 
tinued sterilization— Sterilization  under  pressure— Apparatus  em- 
ployed—Chemical disinfection  and  sterilization  .  .  .  .  .  47-70 


CHAPTER    IV. 

Principles  involved  in  the  methods  of  isolation  of  bacteria  in  pure 
culture  by  the  plate  method  of  Koch— Materials  employed       .       .       71-76 

CHAPTER    V. 

Preparation  of  nutrient  media— Bouillon,  gelatin,  agar-agar,  potato, 
blood-serum,  etc *  77-103 


x  CONTENTS. 

CHAPTER  VI. 

PAGE 

Preparation  of  the  tubes,  flasks,  etc.,  in  which  the  media  are  to 
be  preserved 104-107 

CHAPTEE    VII. 

Technique  of  making  plates— Esmarch  tubes,  Petri  plates,  etc.       .    108-119 

CHAPTEE   VIII. 

The  incubating  oven— Gas-pressure  regulator— Thermo-regulator— 
Safety  burner  employed  in  heating  the  incubator  ....  120-127 

CHAPTEE    IX. 

The  study  of  colonies— Their  naked-eye  peculiarities  and  their  ap- 
pearance under  different  conditions  —  Differences  in  the  structure 
of  colonies  of  different  species  of  bacteria— Stab  cultures— Slant 
cultures  .  .  .  ;  .  .  .  .....  .'  .  .  128-132 

CHAPTEE    X. 

Methods  of  staining— Solutions  employed— Preparation  and  stain- 
ing of  cover-slips—Preparation  of  tissues  for  section-cutting— Stain- 
ing of  tissues— Special  staining  methods 133-169 

CHAPTEE    XI. 

Systematic  study  of  an  organism— Points  to  be  considered  in  iden- 
tifying an  organism  as  a  definite  species  .  .  .  ,  .  .  170-196 

CHAPTEE    XII. 

Inoculation  of  animals— Subcutaneous  inoculation ;  intravenous 
injection— Inoculation  into  the  great  serous  cavities ;  and  into  the  an- 
terior chamber  of  the  eye— Observation  of  animals  after  inoculation  197-218 

CHAPTEE    XIII. 

Post-mortem  examination  of  animals— Bacteriological  examina- 
tion of  the  tissues— Disposal  of  tissues  and  disinfection  of  instru- 
ments after  the  examination  .  .  .  219-224 


CONTENTS.  xi 

APPLICATION    OF    THE    METHODS    OF 
BACTERIOLOGY.      DESCRIPTIONS 
OF  SOME  OF  THE  MORE  IM- 
PORTANT SPECIES. 

CHAPTER  XIV. 

PAGE 

To  obtain  material  with  which  to  begin  work     .       .  "  .  .       .       .    225-228 

CHAPTEE  XV. 

Various  experiments  in  sterilization  by  steam  and  by  hot  air       .    229-233 

CHAPTEE  XVI. 

Suppuration  —  The  staphylococcus  pyogenes  aureus  —  Staphylo- 
coccus  pyogenes  albus  and  citreus— Streptococcus  pyogenes— Bacil- 
lus pyocyanus— General  remarks 234-253 

CHAPTEE  XVII. 

Sputum  septicsemia— Septicaemia  resulting  from  the  presence  of 
the  micrococcus  tetragenus  in  the  tissues— Tuberculosis  .  .  .  254-265 

CHAPTEE    XVIII. 

Tuberculosis— Microscopic  appearance  of  miliary  tubercles— En- 
capsulation of  tuberculous  foci— Diffuse  caseation  —  Cavity-forma- 
tion—Primary  infection— Modes  of  infection— Location  of  the  bacilli 
in  the  tissues— Staining  peculiarities- Organisms  with  which  the 
bacillus  tuberculosis  may  be  confounded— Points  of  differentiation  .  266-286 

CHAPTEE    XIX. 

Glanders — Characteristics  of  the  disease — Histological  structure  of 
the  glanders  nodule — Susceptibility  of  different  animals  to  glanders 
— The  bacillus  of  glanders ;  its  morphological  and  cultural  peculiari- 
ties—Diagnosis of  glanders 287-295 

CHAPTEE  XX. 

Bacillus  diphtheriss—Its  isolation  and  cultivation— Morphological 
and  cultural  peculiarities— Pathogenic  properties —Variations  in 
virulence 296-311 

CHAPTEE    XXL 

Typhoid  fever— Study  of  the  organism  concerned  in  its  produc- 
tion—The bacterium  coli  commune— Its  resemblance  to  the  bacillus 
of  typhoid  fever— Its  morphological,  cultural,  and  pathogenic  prop- 
erties—Its differentiation  from  the  bacillus  typhi  abdominalis  .  .  312-329 


xii  CON1ENTS. 


CHAPTER  XXII. 

PAGE 

The  spirillum  (comma  bacillus)  of  Asiatic  cholera— Its  morphologi- 
cal and  cultural  peculiarities— Pathogenic  properties— The  bacterio- 
logical diagnosis  of  Asiatic  cholera 330-358 

CHAPTER  XXIII. 

Organisms  of  interest,  historically  and  otherwise,  that  have  been 
confounded  with  the  spirillum  of  Asiatic  cholera— Their  peculiari- 
ties and  differential  features— The  vibrio  proteus,  or  bacillus  of  Fink- 
ler  and  Prior— The  spirillum  tyrogenum,  or  cheese  spirillum  of  Deneke 
—The  spirillum  of  Miller— The  vibrio  Metc.hnikovi  ....  359-375 

CHAPTER  XXIV. 

Study  of  the  bacillus  anthracis  and  the  effects  produced  by  its 
inoculation  into  animals— Peculiarities  of  the  organism  under  vary- 
ing conditions  of  surroundings -  376-388 

CHAPTER    XXV. 

The  more  important  of  the  organisms  found  in  the  soil— The  nitri- 
fying bacteria— The  bacillus  of  tetanus— Bacillus  of  malignant 
oedema— Bacillus  of  symptomatic  anthrax 389-413 

CHAPTER  XXVI. 

Infection  and  immunity— The  types  of  infection  ;  intimate  nature 
of  infection — Septicaemia,  Toxsemia,  variations  in  infectious  pro- 
cesses—Immunity, natural  and  acquired— The  hypotheses  that 
have  been  advanced  in  explanation  of  immunity— Conclusions  .  414-438 

CHAPTER  XXVII. 

Bacteriological  study  of  water— Methods  employed— Precautions 
to  be  observed— Apparatus  used,  and  methods  of  using  them— 
Methods  of  investigating  air  and  soil  .  .  .  .  .  .  439-463 

CHAPTER  XXVIII. 

Methods  of  testing  disinfectants  and  antiseptics— Experiments 
illustrating  the  precautions  to  be  taken— Experiments  in  skin  dis- 
infection .  464-476 


APPENDIX. 

Apparatus  necessary  in  a  beginner's  bacteriological  laboratory     .    477-492 


OF   THE 

UNIVERSITY 

OF 

1LIFQR1 


BACTERIOLOGY. 


INTRODUCTION. 

"Omne  vivum  ex  vivo" — The  overthrow  of  the  doctrine  of  spontaneous 
generation— Earlier  bacteriological  studies— The  birth  of  modern  bacteri- 
ology. 

THE  study  of  Bacteriology  may  be  said  to  have  had 
its  beginning  with  the  observations  of  Antony  van 
Leeuwenhoek  in  the  year  1675.  Though  it  is  during 
the  past  decade  and  a  half  that  this  line  of  research  has 
received  its  greatest  impulse,  yet,  by  a  review  of  the 
developmental  stages  through  which  it  has  passed  in  its 
life  of  more  than  two  centuries,  we  see  that  it  has  a 
most  interesting  and  instructive  history.  From  the 
very  outset  its  history  is  inseparably  connected  with 
that  of  medicine,  and  as  it  now  stands  its  relations  to 
hygiene  and  preventive  medicine  are  of  fundamental  im- 
portance. It  is,  indeed,  through  a  more  intimate  ac- 
quaintance with  the  biological  activities  of  the  unicellular, 
vegetable  micro-organisms  that  modern  hygiene  has  at- 
tained the  prominence  and  importance  now  justly  ac- 
corded to  it.  Through  studies  in  the  domain  of  bac- 
teriology our  knowledge  of  the  causation,  course,  and 
prevention  of  infectious  diseases  is  daily  becoming  more 
accurate,  and  it  is  needless  to  emphasize  the  relation 
of  such  knowledge  to  the  manifold  problems  that  pre- 
sent themselves  to  the  sanitarian.  Though  the  contri- 

2 


14  BACTERIOLOGY. 

butions  which  have  done  most  to  place  bacteriology  on 
the  footing  of  a  science  are  those  of  recent  years,  still, 
during  the  earlier  stages  of  its  development,  many  obser- 
vations were  made  which  formed  the  foundation  work 
for  much  that  was  to  follow.  Before  regularly  begin- 
ning our  studies,  therefore,  it  may  be  of  advantage  to 
acquaint  ourselves  with  the  more  prominent  of  these 
investigations. 

Antony  van  Leeuwenhoek,  the  first  to  describe  the 
bodies  now  recognized  as  bacteria,  was  born  at  Delft, 
in  Holland,  in  1632.  He  was  not  considered  a  man  of 
liberal  education,  having  been  during  his  early  years  an 
apprentice  to  a  linendraper.  During  his  apprenticeship 
he  learned  the  art  of  lens-grinding,  in  which  he  became 
so  proficient  that  he  eventually  perfected  a  simple  lens 
by  means  of  which  he  was  enabled  to  see  objects  of  much 
smaller  dimensions  than  any  hitherto  seen  with  the  best 
compound  microscopes  in  existence  at  that  date.  At 
the  time  of  his  discoveries  he  was  following  the  trade 
of  linendraper  in  Amsterdam. 

In  1675  he  published  the  fact  that  he  had  succeeded 
in  perfecting  a  lens  by  means  of  which  he  could  detect 
in  a  drop  of  rain-water  living,  motile  "  animalcules  "  of 
the  most  minute  dimensions — smaller  than  anything  that 
had  hitherto  been  seen.  Encouraged  by  this  discovery, 
he  continued  to  examine  various  substances  for  the 
presence  of  what  he  considered  animal  life  in  its  most 
minute  form.  He  found  in  sea-water,  in  well-water,  in 
the  intestinal  canal  of  frogs  and  birds,  and  in  his  own 
diarrhoaal  evacuations,  objects  that  differentiated  them- 
selves the  one  from  the  other,  not  only  by  their  shape 
and  size,  but  also  by  the  peculiarity  of  movement  which 
some  of  them  were  seen  to  possess.  In  the  year  1683 


INTRODUCTION.  15 

he  discovered  in  the  tartar  scraped  from  between  the 
teeth  a  form  of  micro-organism  upon  which  he  laid 
special  stress.  This  observation  he  embodied  in  the 
form  of  a  contribution  which  was  presented  to  the 
Royal  Society  of  London  on  September  14, 1683.  This 
paper  is  of  particular  importance,  not  only  because  of 
the  careful,  objective  nature  of  the  description  given 
of  the  bodies  seen  by  him,  but  also  for  the  illustrations 
which  accompany  it.  From  a  perusal  of  the  text  and 
an  inspection  of  the  plates  there  remains  little  room  for 
doubt  that  Leeuwenhoek  saw  with  his  primitive  lens 
the  bodies  now  recognized  as  bacteria. 

Upon  seeing  these  bodies  he  was  apparently  very 
much  impressed,  for  he  writes :  "  With  the  greatest 
astonishment  I  observed  that  everywhere  through  the 
material  which  I  was  examining  were  distributed  animal- 
cules of  the  most  microscopic  dimensions,  which  moved 
themselves  about  in  a  remarkably  energetic  way." 

This  discovery  was  shortly  followed  by  others  of 
an  equally  important  nature.  His  field  of  observation 
appears  to  have  increased  rapidly,  for  after  a  time  he 
speaks  of  bodies  of  much  smaller  dimensions  than  those 
at  first  described  by  him. 

Throughout  all  of  Leeuwenhoek's  work  there  is  a 
conspicuous  absence  of  the  speculative.  His  contribu- 
tions are  remarkable  for  their  purely  objective  nature. 

After  the  presence  of  these  organisms  in  water,  in  the 
mouth,  and  in  the  intestinal  evacuations  was  made 
known  to  the  world,  it  is  hardly  surprising  that  they 
were  immediately  seized  upon  as  the  explanation  of  the 
origin  of  many  obscure  diseases.  So  universal  became 
the  belief  in  a  causal  relation  between  these  u  animal- 
cules "  and  disease,  that  it  amounted  almost  to  a  germ 


16  BACTERIOLOGY. 

mania.  It  became  the  fashion  to  suspect  the  presence  of 
these  organisms  in  all  forms  and  kinds  of  disease,  simply 
because  they  had  been  demonstrated  in  the  mouth,  intes- 
tinal evacuations,  and  water. 

Though  nothing  of  value  at  the  time  had  been  done 
in  the  way  of  classification,  and  still  less  in  separating 
and  identifying  the  members  of  this  large  group,  still, 
the  foremost  men  of  the  day  did  not  hesitate  to  ascribe 
to  them  not  only  the  property  of  producing  pathological 
conditions,  but  some  even  went  so  far  as  to  hold  that 
variations  in  the  appearance  of  symptoms  of  disease 
were  the  result  of  differences  in  the  behavior  of  the 
organisms  in  the  tissues. 

Marcus  Antonius  Plenciz,  a  physician  of  Vienna  in 
1762,  declared  himself  a  firm  believer  in  the  work  of 
Leeuwenhoek,  and  based  the  doctrine  which  he  taught 
upon  the  discoveries  of  the  Dutch  observer  and  upon 
observations  of  a  confirmatory  nature  which  he  himself 
had  made.  The  doctrine  of  Plenciz  assumed  a  causal  re- 
lation between  the  micro-organisms  discovered  and  de- 
scribed by  Leeuwenhoek  and  all  infectious  diseases.  He 
claimed  that  the  material  of  infection  could  be  nothing 
else  than  a  living  substance,  and  endeavored  on  these 
grounds  to  explain  the  variations  in  the  period  of  incuba- 
tion of  the  different  infectious  diseases.  He  likewise 
believed  the  living  contagium  to  be  capable  of  multipli- 
cation within  the  body,  and  spoke  of  the  possibility  of 
its  transmission  through  the  air.  He  claimed  a  special 
germ  for  each  disease,  holding  that  just  as  from  a  given 
cereal  only  one  kind  of  grain  can  grow,  so  by  the  special 
germ  for  each  disease  only  that  disease  can  be  produced. 

He  found  in  all  decomposing  matters  innumerable 
minute  "  animalculse,"  and  was  so  firmly  convinced  of 


INTROD  UCTION.  1  7 

their  etiological  relation  to  the  process  that  he  formu- 
lated the  law :  that  decomposition  can  only  take  place 
when  the  decomposable  material  becomes  coated  with  a 
layer  of  the  organisms,  and  can  proceed  only  when  they 
increase  and  multiply. 

However  convincing  the  arguments  of  Plenciz  may 
appear,  they  seem  to  have  been  lost  sight  of  in  the  course 
of  subsequent  events,  and  by  a  few  were  even  regarded 
as  the  productions  of  an  unbalanced  mind.  For  ex- 
ample, as  late  as  1820  we  find  Ozanam  expressing  him- 
self on  the  subject  as  follows  :  "  Many  authors  have 
written  concerning  the  animal  nature  of  the  contagion 
of  infectious  diseases ;  many  have  indeed  assumed  it  to 
be  developed  from  animal  substances  and  that  it  is  itself 
animal,  and  possesses  the  property  of  life ;  I  shall  not 
waste  time  in  efforts  to  refute  these  absurd  hypotheses." 

Similar  expressions  of  opinion  were  heard  from  many 
other  medical  men  of  the  time,  all  tending  in  the  same 
direction,  all  doubting  the  possibility  of  these  micro- 
scopic creatures  belonging  to  the  world  of  living 
things. 

It  was  not  until  between  the  fourth  and  fifth  decade 
of  the  present  century  that  by  the  fortunate  coincidence 
of  a  number  of  important  discoveries  the  true  relation 
of  the  lower  organisms  to  infectious  diseases  was  scien- 
tifically pointed  out.  With  the  investigations  of  Pasteur 
upon  the  cause  of  putrefaction  in  beer  and  the  souring 
of  wine ;  with  the  discovery  by  Pollender  and  Davaine 
of  the  presence  of  rod-shaped  organisms  in  the  blood  of 
all  animals  dead  of  splenic  fever,  and  with  the  progress 
of  knowledge  upon  the  parasitic  nature  of  certain  diseases 
of  plants,  the  old  question  of  "  contagium  animatum  " 
again  began  to  receive  attention.  It  was  taken  up  by 


18  BACTERIOLOGY. 

Heule,  and  it  was  he  who  first  logically  taught  this 
doctrine  of  infection. 

The  main  point,  however,  that  had  occupied  the  atten- 
tion of  scientific  men  from  time  to  time  for  a  period  of 
about  two  hundred  years  subsequent  to  Leeuwenhoek's 
discoveries,  was  the  origin  of  these  bodies.  Do  they 
generate  spontaneously,  or  are  they  the  descendants  of 
pre-existing  creatures  of  the  same  kind  ?  was  the  all- 
important  question.  Among  the  participants  in  this 
discussion  were  many  of  the  most  distinguished  men  of 
the  day. 

In  1749  Needham,  who  held  firmly  to  the  opinion 
that  the  bodies  which  were  creating  such  a  general 
interest  developed  spontaneously,  as  the  result  of  vege- 
tative changes  in  the  substances  in  which  they  were 
found,  attempted  to  demonstrate  by  experiment  the 
grounds  upon  which  he  held  this  view.  He  maintained 
that  the  bacteria  which  were  seen  to  appear  around  a 
grain  of  barley  which  was  allowed  to  germinate  in  a 
watch-crystal  of  water,  which  had  been  carefully  covered, 
were  the  result  of  changes  in  the  barley-grain  itself, 
incidental  to  its  germination. 

Spallanzani,  in  1769,  drew  attention  to  the  laxity  of 
the  methods  employed  by  Needham,  and  demonstrated 
that  if  infusions  of  decomposable  vegetable  matter  were 
placed  in  flasks,  which  were  then  hermetically  sealed, 
and  the  flasks  and  their  contents  allowed  to  remain  for 
some  time  in  a  vessel  of  boiling  water,  neither  living 
organisms  could  be  detected  nor  would  decomposition 
appear  in  the  infusions  so  treated.  The  objection  raised 
by  Treviranus,  viz.,  that  the  high  temperature  to  which 
the  infusions  had  been  subjected  had  so  altered  them 
and  the  air  about  them,  that  the  conditions  favorable  to 


INTRODUCTION.  19 

spontaneous  generation  no  longer  existed,  was  met  by 
Spallanzani  by  gently  tapping  one  of  the  flasks,  that 
had  been  boiled,  against  some  hard  object  until  a  minute 
crack  was  produced  ;  invariably  organisms  and  decompo- 
sition appeared  in  the  flask  thus  treated. 

From  the  time  of  the  experiments  of  Spallanzani 
until  as  late  as  1836  but  little  advance  was  made  in  the 
elucidation  of  this  obscure  problem. 

In  1836  Schulze  attracted  attention  to  the  subject  by 
the  convincing  nature  of  his  investigations.  He  showed 
that  if  the  air  which  gained  access  to  boiled  infusions 
was  robbed  of  its  living  organisms  by  being  caused  to 
pass  through  strong  acid  or  alkaline  solutions  no  de- 
composition appeared,  and  living  organisms  could  not 
be  detected  in  the  infusions.  Following  quickly  upon 
this  contribution  came  Schwann,in  1837,  and  somewhat 
later  (1854)  Schroder  and  Dusch,  with  similar  results 
obtained  by  somewhat  different  means.  Schwann  de- 
prived the  air  which  passed  to  his  infusions  of  its  living 
particles  by  passing  it  through  highly-heated  tubes ; 
whereas  Schroder  and  Dusch,  by  means  of  cotton-wool 
-interposed  between  the  boiled  infusion  and  the  outside 
air,  robbed  the  air  passing  to  the  infusions  of  its  organ- 
isms by  the  simple  process  of  filtration.  In  1860 
Hoffmann  and  in  1861  Chevreul  and  Pasteur  demon- 
strated that  the  precautions  taken  by  the  preceding  in- 
vestigators for  rendering  the  air  which  entered  these 
flasks  free  from  bacteria  were  not  necessary ;  that  all 
that  was  necessary  to  prevent  the  access  of  bacteria  to 
the  infusions  in  the  flasks  was  to  draw  out  the  neck  of 
the  flask  into  a  fine  tube,  bend  it  down  along  the  side 
of  the  flask,  and  then  bend  it  up  again  a  few  inches 
from  its  extremity,  and  leave  the  mouth  open.  The 


20  BACTERIOLOGY. 

infusion  was  then  to  be  boiled  in  the  flask  thus  prepared 
and  the  mouth  of  the  tube  left  open.  The  organisms 
which  now  fall  into  the  tube  will  be  arrested  by  the  drop 
of  water  of  condensation  which  collects  at  its  lowest 
angle,  and  none  can  enter  the  flask. 

Though  from  our  present-day  standpoint  the  results 
of  these  investigations  seem  to  be  of  a  most  convincing 
nature,  yet  there  existed  at  the  time  many  who  required 
additional  proof  that  "  spontaneous  generation  "  was  not 
the  explanation  for  the  mysterious  appearance  of  these 
minute  living  objects.  The  majority,  if  not  all,  of  such 
doubts  were  subsequently  dissipated  through  the  well- 
known  investigations  of  Tyndall  upon  the  floating  matters 
of  the  air.  In  these  studies  he  demonstrated  by  experi- 
ments that  the  presence  of  living  organisms  in  decom- 
posing fluids  was  always  to  be  explained  either  by  the 
pre-existence  of  similar  living  forms  in  the  infusion  or 
upon  the  walls  of  the  vessel  containing  it,  or  by  the 
infusion  having  been  exposed  to  air  which  had  not  been 
deprived  of  its  organisms. 

Throughout  all  the  work  bearing  upon  this  subject, 
from  the  time  of  Spallanzani  to  that  of  Tyndall,  certain 
irregularities  were  constantly  appearing.  It  was  found 
that  particular  substances  required  to  be  heated  for  a 
much  longer  time  than  was  necessary  to  render  other 
substances  free  from  living  organisms,  and  even  under 
the  most  careful  precautions  decomposition  would  occa- 
sionally appear. 

In  1762  Bonnet,  who  was  deeply  interested  in  this 
subject,  suggested,  in  reference  to  the  results  obtained 
by  Needham,  the  possibility  of  the  existence  of  "  germs, 
or  their  eggs,"  which  have  the  power  to  resist  the  tern- 


INTRODUCTION.  21 

perature  to  which  some  of  the  infusions  employed  in 
Needham's  experiments  had  been  subjected. 

More  than  a  hundred  years  after  Bonnet  had  made 
this  purely  speculative  suggestion  it  became  the  task  of 
Ferdinand  Cohn,  of  Breslau,  to  demonstrate  its  accuracy. 

Cohn  repeated  the  foregoing  experiments  with  like 
results.  He  concluded  that  the  irregularities  could  only 
be  due  to  either  the  existence  of  more  resistant  species 
of  bacteria  or  to  more  resistant  stages  into  which  certain 
bacteria  have  the  property  of  passing.  After  much 
work  he  demonstrated  that  certain  of  the  rod-shaped 
organisms  possess  the  power  of  passing  into  a  resting 
or  spore  stage  in  the  course  of  their  life  cycle,  and 
when  in  this  stage  they  are  much  less  susceptible  to  the 
deleterious  action  of  high  temperatures  than  when  they 
are  growing  as  normal  vegetative  forms.  With  the 
discovery  of  these  more  resistant  spores  the  doctrine  of 
spontaneous  generation  received  its  death-blow.  It  was 
no  longer  difficult  to  explain  the  irregularities  in  the 
foregoing  experiments,  nor  was  it  any  longer  to  be 
doubted  that  putrefaction  and  fermentation  were  the 
result  of  bacterial  life  and  not  the  cause  of  it,  and  that 
these  bacteria  were  the  offspring  from  pre-existing 
similar  forms.  In  other  words,  the  law  of  Harvey, 
Omne  vivum  ex  ovo,  or  its  modification,  Omne  vivum  ex 
vivo,  was  shown  to  apply  not  only  to  the  more  highly 
organized  members  of  the  animal  and  vegetable  king- 
doms, but  to  the  most  microscopic,  unicellular  creatures 
as  well. 

The  establishment  of  this  point  served  as  an  impetus 
to  further  investigations,  and  as  the  all-important  ques- 
tion was  that  concerning  the  relation  of  these  micro- 
scopic organisms  to  disease,  attention  naturally  turned 

2* 


22  BACTERIOLOGY. 

into  this  channel  of  study.  Even  before  the  hypothesis 
of  spontaneous  generation  had  received  its  final  refuta- 
tion a  number  of  observations  of  a  most  important 
nature  had  been  made  by  investigators  who  had  long 
since  ceased  to  consider  spontaneous  generation  as  a 
tenable  explanation  of  the  origin  of  the  microscopic 
living  particles. 

In  the  main,  these  studies  had  been  conducted  upon 
wounds  and  the  infections  to  which  they  are  liable ;  in 
fact,  the  evolution  of  our  knowledge  of  bacteriology  to 
the  point  it  now  occupies  is  so  intimately  associated 
with  this  particular  line  of  investigation  that  a  few 
historical  facts  in  connection  with  it  may  not  be  without 
interest. 

The  observations  of  Rindfleisch,  in  1866,  in  which  he 
describes  the  presence  of  small,  pin-head  points  in  the 
myocardium  and  general  musculature  of  individuals 
that  have  died  as  a  result  of  infected  wounds,  offer, 
probably,  the  first  reliable  contribution  to  this  subject. 
He  studied  the  tissue  changes  round  about  these  points 
to  the  stage  of  miliary  abscess  formation.  He  refers 
to  the  organisms  as  "  vibrios."  Almost  simultaneously 
Von  Recklinghausen  and  Waldeyer  described  similar 
changes  that  they  had  observed  in  pyaemia  and  occa- 
sionally secondary  to  typhoid  fever.  Von  Reckling- 
hausen believed  the  granules  seen  in  the  abscess-points 
to  be  micrococci  and  not  tissue  detritus,  and  gave  as 
the  reason  that  they  were  regular  in  size  and  shape,  and 
gave  specific  reactions  with  particular  staining  fluids. 
Birch-Hirschfeld  was  able  to  trace  bacteria  found  in  the 
blood  and  organs  to  the  wound  as  the  point  of  entrance, 
and  believed  both  the  local  and  constitutional  condition 
to  stand  in  direct  ratio  to  the  number  of  spherical 


INTRODUCTION.  23 

bacteria  present  in  the  wound.  He  observed  also  that 
as  the  organisms  increased  in  number  they  could  often 
be  found  within  the  bodies  of  pus  corpuscles.  His 
studies  of  pyaemia  led  him  to  the  important  conclusion 
that  in  this  condition  micro-organisms  were  always 
present  in  the  blood. 

Of  immense  importance  to  the  subject  were  the  in- 
vestigations of  Klebs,  made  at  the  Military  Hospital 
at  Carlsruhe  in  1870-71.  He  not  only  saw,  as  others 
before  him  had  done,  that  bacteria  were  present  in  dis- 
eases following  upon  the  infection  of  wounds,  but 
described  the  manner  in  which  the  organisms  had 
gained  entrance  from  the  point  of  injury  to  the  internal 
organs  and  blood.  His  opinion  was  that  the  spherical 
and  rod-shaped  bodies  that  he  saw  in  the  secretions  of 
wounds  were  closely  allied,  and  gave  to  them  the  desig- 
nation "  microsporon  septicum."  His  opinion  was  that 
the  organisms  gained  access  to  the  tissues  round  about 
the  point  of  injury  both  by  the  aid  of  the  wandering 
leucocytes  and  by  being  forced  through  the  connective- 
tissue  lymph  spaces  by  the  mechanical  pressure  of  mus- 
cular contraction. 

On  erysipelatous  inflammations  secondary  to  injury 
important  investigations  were  also  being  made,  Wilde, 
Orth,  Yon  Recklinghausen,  Lukomsky,  Billroth,  Ehr- 
lich,  Fehleisen,  and  others  agreeing  that  in  these  condi- 
tions micro-organisms  could  always  be  detected  in  the 
lymph  channels  of  the  subcutaneous  tissues;  and 
through  the  work  of  Oertel,  Nassiloff,  Classen,  Letze- 
rich,  Klebs,  and  Eberth  the  constant  presence  of  bacteria 
in  the  diphtheritic  deposits  at  times  seen  on  open  wounds 
was  established. 

Simple   and   natural   as  all   this    may   seem   to   us 


24  BACTERIOLOGY. 

now,  the  stage  to  which  the  subject  had  developed 
when  these  observations  were  recorded  did  not  admit 
of  their  meeting  with  unconditional  acceptance.  The 
only  strong  argument  in  favor  of  the-  etiological  rela- 
tion of  the  organisms  that  had  been  seen,  to  the  diseases 
with  which  they  were  associated,  was  the  constancy 
of  this  association.  No  efforts  had  been  made  to  isolate 
them,  and  few  or  none  to  reproduce  the  pathological 
conditions  by  inoculation.  Moreover,  not  a  small  num- 
ber of  investigators  were  skeptical  as  to  the  impor- 
tance of  these  observations ;  many  claimed  that  micro- 
organisms were  normally  present  in  the  blood  and 
tissues  of  the  body,  aud  some  even  believed  that  the 
organisms  seen  in  the  diseased  conditions  were  the  result 
rather  than  the  cause  of  the  maladies.  It  is  hardly 
necessary  to  do  more  than  say  that  both  of  these  views 
were  purely  speculative,  and  have  never  had  a  single 
reliable  experimental  argument  in  their  favor.  Billroth 
and  Tiegel,  who  held  to  the  former  opinion,  did  endeavor 
to  prove  their  position  through  experimental  means,  but 
the  methods  employed  by  them  were  of  such  an  untrust- 
worthy nature  that  the  fallacy  of  deductions  drawn  from 
them  was  very  quickly  demonstrated  by  subsequent  in- 
vestigators. Their  method  for  demonstrating  the  pres- 
ence of  micro-organisms  in  normal  tissue  was  to  remove 
bits  of  tissue  from  the  healthy  animal  body  with  heated 
instruments  and  drop  them  into  hot  melted  paraffin, 
holding  that  all  living  organisms  on  the  surface  of  the 
tissues  would  be  destroyed  by  the  high  temperature,  and 
that  if  decomposition  should  subsequently  occur  it  would 
prove  that  it  was  the  result  of  the  growth  of  bacteria  in 
the  depths  of  the  tissue  to  which  the  heat  had  not  pene- 
trated. Decomposition  did  usually  set  in,  and  they 


INTRODUCTION.  25 

accepted  this  as  proof  of  the  accuracy  of  their  view. 
Attention  was,  however,  shortly  called  to  the  fact  that 
in  cooling  there  was  contraction  of  the  paraffin  resulting 
usually  in  the  production  of  small  rents  and  cracks  in 
which  dust,  and  bacteria  lodged  upon  it,  could  accumu- 
late and  finally  gain  access  to  the  tissues,  with  the  occur- 
rence of  decomposition  as  a  consequence.  Their  results 
were  thus  explained  after  a  manner  analogous  to  that 
employed  by  Spallanzani,  in  1769,  in  demonstrating  to 
Treviranus  the  fallacy  of  the  opinion  held  by  him  and 
the  accuracy  of  his  own  views,  viz.,  that  it  was  always 
through  the  access  of  organisms  from  without  that  de- 
composition primarily  originates.  (See  page  18.) 

Under  the  most  careful  precautions,  against  which  no 
objection  could  be  raised,  the  experiments  of  Billroth 
and  Tiegel  were  repeated  by  Pasteur,  Burdon-Sanderson, 
and  Klebs,  but  with  failure  in  each  and  every  instance 
to  demonstrate  the  presence  of  bacteria  in  the  healthy 
living  tissues. 

The  fundamental  researches  of  Koch  (1881)  upon 
pathogenic  bacteria  and  their  relation  to  the  infectious 
diseases  of  animals  differed  from  those  of  preceding  in- 
vestigators in  many  important  respects.  The  scientific 
methods  of  analysis  with  which  each  and  eveiy  obscure 
problem  was  met  as  it  arose  served  at  once  to  distinguish 
the  worker  as  a  pioneer  in  this  hitherto  but  partly  culti- 
vated domain.  The  outcome  of  these  experiments  was 
the  establishment  of  a  foundation  upon  which  bacteri- 
ology of  the  future  was  to  rest.  He,  for  the  first  time, 
demonstrated  that  distinct  varieties  of  infection,  as  evi- 
denced by  anatomical  changes,  are  due  in  many  cases  to 
the  activities  of  specific  organisms,  and  that  by  proper 
methods  it  is  possible  to  isolate  these  organisms  in  pure 


26  BACTERIOLOGY. 

culture,  to  cultivate  them  indefinitely,  to  reproduce  the 
conditions  by  inoculation  of  these  pure  cultures  into 
susceptible  animals,  aud,  by  continuous  inoculation  from 
an  infected  to  a  healthy  animal,  to  continue  the  disease 
at  will.  By  the  methods  that  he  employed  he  demon- 
strated a  series  of  separate  and  distinct  diseases  that  can 
be  produced  in  mice  and  rabbits  by  the  injection  into 
their  tissues  of  putrid  substances.  The  disease  known 
as  septicaemia  of  mice ;  also  a  disease  characterized  by 
progressive  abscess  formation ;  and  pyaemia  and  septi- 
ca3mia  of  rabbits,  are  among  the  affections  produced  by 
him  in  this  way.  It  was  in  the  course  of  this  work  that 
the  Abbe  system  of  substage  condensing  apparatus  was 
first  used  in  bacteriology  ;  that  the  aniline  dyes  suggested 
by  Weigert  were  brought  into  general  use ;  that  the 
isolation  and  cultivation  of  bacteria  in  pure  culture  on 
solid  media  was  shown  to  be  possible ;  and  that  animals 
were  employed  as  a  means  of  obtaining  from  mixtures 
pure  cultures  of  pathogenic  bacteria. 

With  the  bounteous  harvest  of  original  and  important 
suggestions  that  was  reaped  from  Koch's  classical  series 
of  investigations  bacteriology  reached  an  epoch  in  its 
development,  and  at  this  period  modern  bacteriology 
may  justly  be  said  to  have  had  its  birth. 

NOTE. — I  have  presented  only  the  most  prominent 
investigations  that  will  serve  to  indicate  the  lines  along 
which  the  subject  has  developed.  For  a  more  detailed 
account  of  the  historical  development  of  the  work  the 
reader  is  referred  to  Loeffler's  Vorlesungen  uber  die  ges- 
chichtliche  Entwickelung  der  Lehre  von  den  Bacterien, 
upon  which  I  have  drawn  largely  in  preparing  the  fore- 
going sketch. 


CHAPTER   I. 

Definition  of  bacteria — Their  place  in  nature — Difference  between  parasites 
and  saprophytes — Nutrition  of  bacteria — Products  of  bacteria — Their  relation 
to  oxygen — Influence  of  temperature  upon  their  growth. 

BY  the  term  bacteria  is  understood  that  large  group 
of  minute  vegetable  organisms  which  multiplies  by  a 
process  of  transverse  division.  They  are  spherical,  oval, 
rod-like,  and  spiral  in  shape,  and  are  commonly  devoid 
of  chlorophyll.1  Owing  to  the  absence  of  chlorophyll 
from  their  composition,  the  bacteria  are  forced  to  obtain 
their  nutritive  materials  from  organic  matters  as  such, 
and  lead,  therefore,  either  a  saprophytic2  or  parasitic3 
form  of  existence. 

Their  life  processes  are  so  rapid  and  energetic  that 
they  result  in  the  most  profound  alterations  in  the 
structure  and  composition  of  the  materials  in  and  upon 
which  they  are  developing. 

Decomposition,  putrefaction,  and  fermentation  result 
from  the  activities  of  the  saprophytio  bacteria,  while 
the  changes  brought  about  in  the  tissues  of  their  host 


1  Chlorophyll  is  the  green  coloring  matter  possessed  by  the  higher  plants 
by  means  of  which  they  are  enabled  in  the  presence  of  sunlight  to  decom- 
pose carbonic  acid  (CO2)  and  ammonia  (NH3)   into  their  elementary  con- 
stituents. 

2  A  saprophyte  is  an  organism  that  obtains  its  nutrition  from  dead  organic 
matter. 

3  A  parasite  lives  always  at  the  expense  of  some  other  living,  organic  crea- 
ture, known  as  its  host,  and  in  the  strictest  sense  of  the  word  cannot  develop 
upon  dead  matter.    There  is,  however,  a  group  of  so-called  "facultative" 
saprophytes  and  parasites  which  possess  the  power  of  accommodating  them- 
selves to  existing  surroundings— at  one  time  leading  a  parasitic,  at  another 
time  a  saprophytic  form  of  existence. 


28  BACTERIOLOGY. 

by  the  pure  parasitic  forms  find  expression  in  disease 
processes  and  not  infrequently  complete  death. 

The  role  played  in  nature  by  the  saprophytic  bacteria 
is  a  very  important  one.  Through  their  presence  the 
highly  complicated  tissues  of  dead  animals  and  vegeta- 
bles are  resolved  into  the  simpler  compounds,  carbonic 
acid,  water,  and  ammonia,  in  which  form  they  may  be 
taken  up  and  appropriated  as  nutrition  by  the  more 
highly  organized  members  of  the  vegetable  kingdom. 
It  is  through  this  ultimate  production  of  carbonic  acid, 
ammonia,  and  water  by  the  bacteria,  as  end-products 
in  the  processes  of  decomposition  and  fermentation  of 
the  dead  animal  and  vegetable  tissues,  that  the  de- 
mands of  growing  vegetation  for  these  compounds  are 
supplied. 

The  chlorophyll  plants  do  not  possess  the  power  of 
obtaining  their  carbon  and  nitrogen  from  such  highly 
organized  and  complicated  substances  as  serve  for  the 
nutrition  of  bacteria,  and  as  the  production  of  these 
simpler  compounds  (CO2,  NH3,  H20)  by  the  animal 
world  is  not  sufficient  to  meet  the  demands  of  the  chlo- 
rophyll plants,  the  importance  of  the  part  played  by 
bacteria  in  making  up  this  deficit  cannot  be  overesti- 
mated. Were  it  not  for  the  activity  of  these  micro- 
scopic living  particles,  all  life  upon  the  surface  of  the 
earth  would  undoubtedly  cease.  Deprive  higher  vegeta- 
tion of  the  carbon  and  nitrogen  supplied  to  it  as  a  result 
of  bacterial  activity,  and  its  development  comes  rapidly 
to  an  end  ;  rob  the  animal  kingdom  of  the  food-stuffs 
supplied  to  it  by  the  vegetable  world,  and  life  is  no 
longer  possible. 

It  is  plain,  therefore,  that  the  saprophytes,  which 
represent  the  large  majority  of  all  bacteria,  must  be 


THEIR  PLACE  IN  NATURE.  29 

looked  upon  by  us  in  the  light  of  benefactors,  without 
which  existence  would  be  impossible. 

With  the  parasites,  on  the  other  hand,  the  conditions 
are  far  from  analogous.  Through  their  activities  there 
is  constantly  a  loss,  rather  than  a  gain,  to  both  the 
animal  and  vegetable  kingdoms.  Their  host  must 
always  be  a  living  body  in  which  exist  conditions  favor- 
able to  their  development,  and  from  which  they  appro- 
priate substances  that  are  necessary  to  the  health  and 
life  of  the  organism  to  which  they  may  have  found 
access ;  at  the  same  time  they  eliminate  substances  as 
products  of  their  nutrition  that  are  directly  poisonous 
to  the  tissues  in  which  they  are  growing. 

In  their  relations  to  humanity,  the  positions  occupied 
by  the  two  biologically  different  groups,  the  saprophytes 
on  the  one  hand  and  the  parasites  on  the  other,  are 
diametrically  opposite : — the  saprophytic  forms  stand 
in  the  relation  of  benefactors,  in  resolving  dead  animal 
and  vegetable  bodies  into  their  component  parts,  which 
serve  as  food  for  living  vegetation,  and,  at  the  same 
time,  they  remove  from  the  surface  of  the  earth  the  re- 
mains of  all  dead  organic  substances  ;  while  the  parasitic 
group  exists  only  at  the  expense  of  the  more  highly 
organized  members  of  both  kingdoms.  It  is  to  the 
parasitic  group  that  the  pathogenic1  organisms  belong. 

In  addition  to  the  saprophytes  that  are  concerned 
in  the  changes  to  which  allusion  has  just  been  made, 
there  exist  other  saprophytic  forms  whose  life  processes 
result  in  specific  changes  of  most  interesting  and  im- 
portant natures.  Some  of  these  are  characterized  by 
their  property  of  producing  pigments  of  different  color; 

1  Pathogenic  organisms  are  those  which  possess  the  property  of  producing 
disease. 


30  BACTERIOLOGY. 

these  are  known  as  the  chromogenic1  forms.  Just  what 
their  exact  role  in  nature  is  it  is  difficult  to  say ;  but  it 
is  probable  that  in  addition  to  their  most  conspicuous 
function  of  color  production,  they  are  also  in  some 
way  concerned  in  the  great  process  of  disintegration 
which  is  constantly  going  on  in  all  dead  organic  sub- 
stances. 

Others,  the  so-called  photogenic,  or  phosphorescent 
bacteria,  possess  the  property  of  producing  light  or  of 
illuminating  the  medium  on  which  they  grow  by  a 
peculiar  phosphorescence.  These  are  found  in  sea- water 
and  in  decomposing  phosphorescent  fish  and  meat. 

Still  others,  the  so-called  zymogenic  bacteria,  are  con- 
cerned in  the  various  fermentations,  while  the  putrefac- 
tive or  saprogenic  bacteria  are  those  that  produce  the 
particular  fermentation  that  we  know  as  putrefaction. 
Another  very  important  saprophytic  group  comprises 
the  so-called  nitrifying  and  denitrifying  bacteria,  whose 
activities  result  in  specific  forms  of  fermentation — the 
former  oxidizing  ammonia  to  nitrous  and  nitric  acid, 
the  latter  reducing  nitric  acid  to  nitrous  acid  and  am- 
monia. The  so-called  thiogenic  bacteria  convert  sul- 
phuretted hydrogen  into  higher  sulphur  compounds. 

We  have  said  that  through  the  agency  of  chlorophyll, 
in  the  presence  of  sunlight,  the  green  plants  are  enabled 
to  obtain  the  amount  of  nitrogen  and  carbon  which  is 
necessary  to  their  growth  from  such  simple  bodies  as 
carbon  dioxide  and  ammonia,  which  they  decompose 
into  their  elementary  constituents.  The  bacteria,  on  the 
other  hand,  owing  to  the  absence  of  chlorophyll  from 
their  tissues,  do  not  possess  this  power.  They  must, 

1  Chromogenic :— possessing  the  property  of  generating  color. 


NUTRITION  OF  BACTERIA.  31 

therefore,  have  their  carbon  and  nitrogen  presented  as 
such,  in  the  form  of  decomposable  organic  substances. 

In  general,  the  bacteria  obtain  their  nitrogen  most 
readily  from  soluble  albumins,  and,  to  a  certain  degree, 
but  by  no  means  so  easily,  from  salts  of  ammonia.  In 
some  of  Nageli's  experiments  it  appeared  probable  that 
they  could  obtain  the  necessary  amount  of  nitrogen 
from  salts  of  nitric  acid.  At  all  events,  he  was  able 
in  certain  cases  to  demonstrate  a  reduction  of  nitric  to 
nitrous  acid,  and  ultimately  to  ammonia.  Nevertheless, 
in  all  of  these  experiments  circumstances  point  to  the 
probability  that  the  nitrogen  obtaiued  by  the  bacteria 
for  building  up  their  tissues  in  the  course  of  their 
development,  was  derived  from  some  source  other  than 
that  of  the  nitric  acid  or  the  nitrates,  and  that  the 
reduction  of  this  acid  was  most  probably  a  secondary 
phenomenon.  It  must  be  borne  in  mind,  however,  that 
there  exists  a  specific  group  of  bacteria,  the  nitrifying 
bacteria,  that  apparently  increase  and  multiply  without 
having  access  to  proteid  .nutrition.  They  are  concerned 
in  the  particular  form  of  fermentation  that  results  in  the 
oxidation  of  ammonia  to  nitrous  and  nitric  acids,  a  pro- 
cess everywhere  in  progress  in  the  superficial  layers  of 
the  soil. 

For  the  supply  of  carbon,  many  of  the  carbon  com- 
pounds serve  as  sources  upon  which  the  bacteria  can 
draw.  The  carbon  deficit,  for  example,  can  be  obtained 
from  sugar  and  bodies  of  like  composition ;  from  glyce- 
rine and  many  of  the  fatty  acids;  and  from  the  alkaline 
salts  of  tartaric,  citric,  malic,  lactic,  and  acetic  acids. 
In  some  instances  carbon  compounds,  which  when 
present  in  concentrated  form  inhibit  the  growth  of  bac- 
teria, may,  when  highly  diluted,  serve  as  nutrition  for 


32  BACTERIOLOGY. 

them.     Salicylic  acid  and  ethyl  alcohol  come  under  this 
head. 

In  addition  to  carbon  and  nitrogen,  water  is  essential 
to  the  life  and  development  of  bacteria.  Without  it 
no  development  occurs,  and  in  many  cases  drying  the 
organisms  results  in  their  death.  Certain  forms,  on  the 
contrary,  though  incapable  of  multiplying  when  in  the 
dry  stage,  may  be  completely  deprived  of  their  water 
without  causing  them  to  lose  the  power  of  reproduction 
when  favorable  conditions  reappear. 

The  closer  study  of  the  bacteria,  and  a  more  intimate 
acquaintance  with  their  nutritive  changes,  demonstrate 
an  appreciable  variability  in  the  character  of  the  sub- 
stances best  suited  for  the  nutrition  of  different  species, 
one  requiring  a  tolerably  concentrated  form  of  nutri- 
tion, while  another  needs  but  a  very  limited  amount  of 
proteid  substance  for  its  development.  Certain  mem- 
bers bring  about  most  profound  alterations  in  the  media 
in  which  they  exist,  while  others  produce  but  little 
apparent  change.  In  one  case  alterations  in  the  reac- 
tion of  the  media  will  be  conspicuous,  while  in  another 
no  such  variation  can  be  detected.  With  the  growth 
of  some  forms  products  resulting  from  processes  of 
fermentation  appear.  Other  varieties  produce  poisons 
of  remarkable  degrees  of  toxicity,  while  the  growth  of 
others  may  be  accompanied  by  the  bodies  characteristic 
of  putrefaction. 

For  the  normal  development  of  bacteria  it  is  not  only 
essential  that  the  sources  from  which  they  can  obtain 
the  necessary  nutritive  elements  should  exist,  but  account 
must  also  be  taken  of  the  products  of  growth  of  the 
organisms  in  these  substances.  Nitrogen  and  carbon 
compounds  in  the  proper  form  to  be  taken  up  and 


NUTRITION  OF  BACTERIA.  33 

appropriated  by  the  organism  may  exist  in  sufficient 
quantities,  and  still  the  growth  of  the  organism  after  a 
very  short  time  be  entirely  checked,  owing  to  the  pro- 
duction during  their  growth  of  substances  inhibitory  to 
their  further  development.  Most  conspicuous  are  the 
changes  produced  by  growing  bacteria  in  the  chemical 
reaction  of  the  media.  Since  the  majority  of  them  grow 
best  in  media  of  a  neutral  or  very  slightly  alkaline 
reaction,  any  excessive  production  of  alkalinity  or 
acidity,  as  a  product  of  growth,  arrests  development^ 
and  no  evidence  of  life  or  further  multiplication  can  be 
detected  until  this  deviation  from  the  neutral  reaction 
has  been  corrected. 

Most  favorable  for  the  development  of  bacteria  are 
neutral  or  very  slightly  alkaline  solutions  of  albumin  in 
one  form  or  another. 

Of  considerable  importance  and  interest  in  the  study 
of  the  nutritive  changes  of  bacteria  is  the  difference  in 
their  relation  to  oxygen.  With  certain  forms  oxygen 
is  essential  for  the  proper  performance  of  their  func- 
tions, while  with  another  group  no  evidence  of  life  can 
be  detected  under  the  access  of  oxygen,  and  in  a  third 
group  oxygen  appears  to  play  but  an  unimportant  part, 
for  development  occurs  as  well  with  as  without  it.  It 
was  Pasteur  who  first  demonstrated  the  existence  of 
species  in  the  bacteria  family  which  not  only  grow  and 
multiply  and  perform  definite  physiological  functions 
without  the  aid  of  oxygen,  but  to  the  existence  of 
which  oxygen  is  positively  harmful.  To  these  he  gave 
the  name  anaerobic  bacteria,  in  contradistinction  to 
another  group  for  the  proper  performance  of  whose 
functions  oxygen  is  essential ;  these  he  called  aerobic 
bacteria.  In  addition  to  these,  there  is  a  third  group 


34  BACTERIOLOGY. 

for  the  maintenance  of  whose  existence  the  absence  or 
presence  of  oxygen  is  apparently  of  no  moment — their 
development  progresses  as  well  with  as  without  it ;  these 
constitute  the  class  known  as  facultative  in  their  relation 
to  this  gas.  It  is  in  this  third  group,  the  facultative, 
that  the  majority  of  bacteria  belong.  Though  the  mul- 
tiplication of  the  facultative  varieties  is  not  interfered 
with  by  either  the  presence  or  absence  of  oxygen,  yet 
experiments  demonstrate  that  the  products  of  their 
growth  are  different  under  the  varying  conditions  of 
absence  or  presence  of  this  gas. 

For  example :  in  the  case  of  certain  of  the  chromo- 
genic  forms  the  presence  or  absence  of  oxygen  has  a 
very  decided  effect  upon  the  production  of  the  pigments 
by  which  they  are  characterized. 

NOTE. — Observe  the  difference  between  the  intensity 
of  color  produced  upon  the  surface  of  the  medium  and 
that  along  the  track  of  the  needle  in  stab-cultures  of 
the  bacillus  prodigiosus  and  of  the  spirillum  rubrum. 
With  the  former  the  red  color  is  apparently  a  product 
dependent  upon  the  presence  of  oxygen,  while  in  the 
latter  the  greatest  intensity  of  color  occurs  at  the  point 
farthest  removed  from  the  action  of  oxygen. 

Another  element  which  plays  a  highly  important  part 
in  the  biological  functions  of  these  organisms  is  the 
temperature  under  which  they  exist.  The  extremes  of 
temperature  under  which  the  majority  of  bacteria  are 
known  to  grow  range  from  5.5°  C.  to  43°  C.  At  the 
former  temperature  development  is  hardly  appreciable ; 
it  becomes  more  and  more  active  until  38°  C.  is  reached, 
when  it  is  at  its  optimum,  and,  as  a  rule,  ceases  with  43° 


GROWTH  AND  DEVELOPMENT  OF  BACTERIA.      35 

C. ;  though  species  exist  that  will  multiply  at  as  high  a 
temperature  as  70°  C.  and  others  as  low  as  0°  C.  The 
studies  of  Globig1,  Miquel2,  and  Macfadyen  and  Bloxall3 
have  demonstrated  that  there  exist  in  the  soil,  in  water, 
in  faBces,  in  sewage,  in  dust,  and,  in  fact,  practically 
everywhere,  bacteria  that  under  artificial  cultivation  show 
no  evidence  of  life  at  a  temperature  lower  than  60°  to 
65°  C.,  and  would  even  grow  at  as  high  a  temperature 
as  70°  to  75°  C.,  degrees  of  heat  sufficient  for  the  coag- 
ulation of  albumin.  Rabinowitsch4  has  likewise  de- 
scribed a  number  of  species  of  these  "  thermophilic " 
bacteria,  as  they  are  called,  but  states  that  it  was  possible 
in  her  experiments  to  obtain  evidence  of  their  growth  at 
a  lower  temperature  (34°  C.  to  44°  C.)  as  well  as  at  the 
higher  temperature  mentioned  by  preceding  investiga- 
tors. The  most  favorable  temperature  for  the  develop- 
ment of  pathogenic  bacteria  is  that  of  the  human  body, 
viz.,  37.5°  C.  There  are  a  number  of  bacteria  com- 
monly present  in  water,  the  so-called  normal  water  bac- 
teria, that  grow  best  at  about  20°  C. 

In  general  then,  from  what  has  been  learned,  it  may 
be  said  that  for  the  growth  and  development  of  bacteria 
organic  matter  of  a  neutral  or  slightly  alkaline  reac- 
tion, in  the  presence  of  moisture  and  at  a  suitable  tem- 
perature, is  necessary.  From  this  can  be  formed  some 
idea  of  the  omnipresence  in  nature  of  these  minute 
vegetable  forms.  Everywhere  that  these  conditions 
obtain  bacteria  can  be  found. 

1  Globig :  Zeitschrift  fur  Hygiene,  Bd.  iii.  S.  294. 

2  Miquel :  Annales  de  Micrograph^,  1888,  pp.  4  to  10. 

3  Macfadyen  and  Bloxall :  Journal  of  Path,  and  Bact.,  vol.  iii.  Part  I. 

4  Rabinowitsch  :  Zeitschrift  fur  Hygiene  u.  Infectionskrankheiten,  Bd.  xx. 
Heft  1,  S.  154  to  164. 


Of   THE 

UNIVERSITY 

Of 


CHAPTER    II. 


Morphology1  of  bacteria— Grouping— Mode  of  multiplication— Spore-forma- 
tion-Motility. 


IN  structure  the  bacteria  are  unicellular,  and  are  seen 
to  exist  as  spherical,  rod-  or  spiral-shaped  bodies. 
They  always  develop  from  pre-existing  cells  of  the 
same  character  and  never  appear  spontaneously. 

The  classifications  of  the  older  authors  and  of  the 
botanists  are  usually  upon  purely  morphological  pecu- 
liarities, and,  because  of  slight  variations  that  are  seen 
to  occur  in  the  size  and  shape  of  one  and  the  same  spe- 
cies, are  more  or  less  complicated.  The  present  tendency 
is  to  simplify  this  morphological  classification,  and  to 
bring  the  bacteria  into  three  great  groups,  with  their 
subdivisions,  the  members  of  each  group  being  deter- 
mined by  their  individual  outline,  viz.,  that  of  a  sphere, 
a  rod,  or  a  spiral. 

To  these  three  grand  divisions  are  given  the  names 
cocci  or  micrococci,  bacilli,  and  spirilla. 

In  the  group  micrococci  belong  all  spherical  forms, 
L  e.,  all  those  forms  the  isolated  individual  members  of 
which  are  of  the  same  diameter  in  all  directions.  (See 
Fig.  1,  a  b  c  d.) 

The  bacilli  comprise  all  oval  or  rod-formed  bacteria. 
(See  Fig.  2.) 

To  the  spirilla  belong  all  organisms  that  are  curved 

1  Morphology  :— Pertaining  to  shape ;  outline. 


GROUPING. 


37 


when  seen  in  short  segments,  or  when  in  longer  threads 
are  twisted  in  the  form  of  a  corkscrew.     (See  Fig.  3.) 


FIG.  l. 


a.  Staphylococci.    6.  Streptococci,     c.  Diplococci.    d.  Tetrads,    e.  Sarcinse. 

FIG.  2. 


xV 


-\ri 


a.  Bacilli  in  pairs,    ft.  Single  bacilli,    c  and  d.  Bacilli  in  threads,    eand/, 
Bacilli  of  variable  morphology. 

FIG.  3.  / 


-TV*" 

^Pw- 


a  6  c  ^ 

a  and  d.  Spirilla  in  short  segments  and  longer  threads— the  so-called 
comma  forms  and  spirals.  6.  The  forms  known  as  spirochetse.  c.  The  thick 
spirals  sometimes  known  as  vibrios. 

3 


38  BACTERIOLOGY. 

The  micrococci  are  subdivided  according  to  their 
grouping,  as  seen  in  growing  cultures,  into  staphylococci 
— those  growing  in  masses  like  clusters  of  grapes  (see 
Fig.  1,  a) ;  streptococci — those  growing  in  chains  con- 
sisting of  a  number  of  individual  cells  strung  together 
like  beads  or  pearls  upon  a  string  (see  Fig.  1,6); 
diplococci — those  growing  in  pairs  (Fig.  1,  o);  tetrads 
— those  developing  as  fours  (Fig.  1,  d) ;  and  sarcince — 
those  dividing  into  fours,  eights,  etc.,  as  cubes — that  is, 
in  centra-distinction  to  all  other  forms,  the  segmenta- 
tion, which  is  rarely  complete,  takes  place  in  three 
directions  of  space,  so  that  when  growing  the  bundle  of 
segmenting  cells  presents  somewhat  the  appearance  of 
a  bale  of  cotton  (Fig.  1,  e). 

To  the  bacilli  belong  all  straight,  rod-shaped  bacteria, 
i.  e.,  those  in  which  one  diameter  is  always  greater  than 
the  other. 

FIG.  4. 


»*" 

a  5  c  d 

a.  Bacillus  subtilis  with  spores,    b.  Bacillus  anthracis  with  spores,    c.  Clos- 
tridium  form  with  spores,    d.  Bacillus  of  tetanus  with  end  spores. 

In  this  group  are  found  those  organisms  the  life  cycle 
of  many  of  which  presents  deviations  from  the  simple 
rod  shape.  Many  of  them  in  the  course  of  development 
increase  in  length  into  long  threads,  along  the  course  of 
which  traces  of  segmentation  may  usually  be  found — 
the  anthrax  bacillus  and  bacillus  subtilis  are  conspicuous 
examples  of  this.  Again,  under  certain  conditions, 
many  of  them  possess  the  property  of  forming  within 


GROUPING.  39 

the  body  of  the  rods  oval,  glistening  spores  (see  Fig.  4), 
and,  if  the  conditions  are  not  altered,  the  rods  may 
entirely  disappear,  so  that  nothing  be  left  in  the  cul- 
ture but  these  oval  spores.  In  some  of  them  this 
phenomenon  of  spore-formation  is  accompanied  by  an 
enlargement  or  swelling  of  the  bacillus  at  the  point  at 
which  the  spore  is  located  (see  Fig.  4,  c  and  d).  Again, 
many  of  them,  from  unfavorable  conditions  of  nutrition, 
aeration,  or  temperature  undergo  pathological  changes 
— that  is,  the  individuals  themselves  experience  altera- 
tions in  their  protoplasm  which  result  in  distortion  of 
their  outline,  and  the  appearance  of  the  so-called  "  in- 
volution forms."  (See  Fig.  5,  a  and  6.)  In  all  of 

FIG.  5. 
'        ft 


A  i  * 

;//   < 


*  s 

a  b 

a.  Spirillum  of  Asiatic  cholera  (comma  bacillus).    &.  Involution  forms  of 
this  organism  as  seen  in  old  cultures. 

these  conditions,  however,  so  long  as  death  has  not 
actually  occurred,  it  is  possible  under  favorable  condi- 
tions to  cause  these  forms  to  revert  to  the  rod-shaped 
ones  from  which  they  originated. 

It  must  be  borne  in  mind,  though,  that  it  is  never 
possible  by  any  means  to  bring  about  changes  in  these 
organisms  that  will  result  in  the  permanent  conversion 
of  the  morphology  of  the  members  of  one  group  into 
that  of  another — that  is,  one  can  never  produce  bacilli 
from  micrococci  or  vice  versa,  and  any  evidence  which 
may  be  presented  to  the  contrary  is  based  upon  untrust- 
worthy methods  of  observation. 


40  BACTERIOLOGY. 

Not  infrequently  bacteria  may  be  observed  irregu- 
larly massed  together  as  a  pellicle.  When  in  this  condi- 
tion they  are  held  together  by  a  gelatinous  material,  and 
are  known  as  zoogloea  of  bacteria.  (See  Fig.  6.) 

Very  short  oval  bacilli  may  sometimes  be  mistaken 
for  micrococci,  and  at  times  micrococci  in  the  stage  of 
segmentation  into  diplococci  may  be  mistaken  for  short 
bacilli ;  but  by  careful  inspection  it  will  always  be 
possible  to  detect  a  continuous  outline  along  the  sides  of 
the  former,  and  a  slight  transverse  indentation  or  par- 
tition-formation between  the  segments  of  the  latter. 
The  high  index  of  refraction  of  spores,  the  property 

FIG.  G. 


Zooglcea  of  bacilli. 

which  gives  to  them  their  glistening  appearance,  will 
always  serve  to  distinguish  them  from  micrococci.  This 
difference  in  refraction  is  especially  noticeable  if  the 
illumination  from  the  reflector  of  the  microscope  with 
which  they  are  examined  be  reduced  to  the  smallest 
possible  bundle  of  light-rays.  The  spores,  moreover, 
take  up  the  staining  reagents  much  less  readily  than  do 
the  micrococci.  The  most  reliable  differential  point,  how- 
ever, is  the  property,  possessed  by  the  spores,  of  develop- 
ing into  bacilli;  and  by  the  spherical  organism  with 
which  it  has  been  confounded,  of  producing  other  micro- 
cocci  of  the  same  round  form. 

For    convenience,  a   common    classification    of   the 


GERMINATION.  41 

bacilli  is  that  based  upon  constant  characteristics  which 
are  seen  to  appear  in  the  course  of  their  development 
under  special  conditions — certain  of  them  possessing  the 
power  of  forming  spores,  while  from  others  this  pecu- 
liarity is  absent. 

As  yet  but  little  is  known  of  the  life  history  of  the 
spiral  forms.  Eiforts  toward  their  cultivation  under 
artificial  conditions  have  thus  far  been  successful  in 
only  a  few  cases.  Morphologically,  they  are  thread-  or 
rod-like  bodies  which  are  twisted  into  the  form  of  spirals. 
In  some  of  them  the  turns  of  the  spiral  are  long,  in 
others  quite  short.  They  are  motile,  and  multiply  ap- 
parently by  the  simple  process  of  fission.1 

The  micrococci  develop  by  simple  fission.  When 
development  is  in  progress  a  single  cell  will  be  seen  to 
elongate  slightly  in  one  of  its  diameters.  Over  the 
centre  of  the  long  axis  thus  formed,  will  appear  a  slight 
indentation  in  the  outer  envelope  of  the  cell ;  this  inden- 
tation will  increase  in  extent  until  there  exist  eventu- 
ally two  individuals  which  are  distinctly  spherical,  as 
was  the  parent  from  which  they  sprang,  or  they  will 
remain  together  for  a  time  as  diplococci.  The  sur- 
faces now  in  juxtaposition  are  flattened  against  one 
another,  and  not  infrequently,  a  fine,  pale  dividing  line 
may  be  seen  between  the  two  cells.  (See  Fig.  1,  c 
and  d.)  A  similar  division  in  the  other  direction  will 
now  result  in  the  formation  of  a  group  of  forms  as 
tetrads. 

In  the  formation  of  staphylococci  such  division  occurs 
irregularly  in  all  directions,  resulting  in  the  produc- 
tion of  the  clusters  in  which  these  organisms  are  com- 

1  Dividing  into  two  transversely. 


42  BACTERIOLOGY. 

monly  seen.  (See  Fig.  1,  a.)  With  the  streptococci, 
however,  the  tendency  is  for  the  segmentation  to  con- 
tinue in  one  direction  only,  resulting  in  the  production 
of  long  chains  of  4,  8,  and  12  individuals.  (See  Fig. 
1,6.) 

The  sarcinse  divide  more  or  less  regularly  in  three 
directions  of  space,  but  instead  of  becoming  separated 
the  one  from  the  other  as  single  cells,  the  tendency  is 
for  the  segmentation  to  be  incomplete ;  the  cells  remain- 
ing together  in  masses.  The  indentations  upon  these 
masses  or  cubes,  which  indicate  the  point  of  incomplete 
fission,  give  to  these  bundles  of  cells  the  appearance  com- 
monly ascribed  to  them — that  of  a  bale  of  cotton  or  a 
packet  of  rags.  (See  Fig.  1,  e.) 

The  multiplication  of  bacilli  is  in  the  main  similar 
to  that  given  for  the  micrococci.  A  dividing  cell  will 
elongate  slightly  in  the  direction  of  its  long  axis ;  an 
indentation  will  appear  about  midway  between  its  poles, 
and  will  become  deeper  and  deeper  until  eventually  two 
daughter  cells  will  be  formed.  This  process  may  occur 
in  such  a  way  that  the  two  young  bacilli  will  adhere 
together  by  their  adjacent  ends  in  much  the  same  way 
that  sausages  are  seen  to  be  held  together  in  strings 
(Fig.  2,  /),  or  the  segmentation  may  take  place  more  at 
right  angles  to  the  long  axis,  so  that  the  proximal  ends 
of  the  young  cells  are  flattened  while  the  distal  extremi- 
ties may  be  rounded  or  slightly  pointed  (Fig.  2,  e). 
The  segmentation  of  the  anthrax  bacillus,  with  which 
we  are  subsequently  to  become  acquainted,  results,  when 
completed,  in  an  indentation  of  the  adjacent  extrem- 
ities of  the  young  segments,  so  that  by  the  aid  of  high 
magnifying  powers  these  surfaces  are  seen  to  be  actually 
concave.  Bacilli  never  divide  longitudinally. 


SPORE-FORMATION.  43 

With  the  spore-forming  bacilli,  under  favorable  con- 
ditions of  nutrition  and  temperature,  the  same  is  seen 
to  occur  daring  vegetation,  but  as  soon  as  these  condi- 
tions become  altered,  either  by  the  exhaustion  of  nutri- 
tion, the  presence  of  detrimental  substances,  unfavorable 
temperatures,  etc.,  there  appears  the  stage  in  their  life 
cycle  to  which  we  have  referred  as  "  spore-formation." 
This  is  the  process  by  which  the  organisms  are  enabled 
to  enter  a  stage  in  which  they  resist  deleterious  influences 
to  a  much  higher  degree  than  is  possible  for  them  when 
in  the  growing  or  vegetative  condition. 

In  the  spore,  resting,  or  permanent  stage,  as  it  is 
called,  no  evidence  of  life  whatever  is  given  by  the 
spores,  though  as  soon  as  the  conditions  which  favor 
their  germination  have  been  renewed,  these  spores  de- 
velop again  into  the  same  kind  of  cells  as  those  from 
which  they  originated,  and  the  appearances  observed  in 
the  vegetative  or  growing  stage  of  their  history  are 
repeated. 

Multiplication  of  spores,  as  such,  does  not  occur; 
they  possess  the  power  of  developing  into  individual 
rods  of  the  same  nature  as  those  from  which  they  were 
formed,  but  not  of  giving  rise  to  a  direct  reproduction  of 
spores. 

When  the  conditions  which  favor  spore-formation 
present,  the  protoplasm  of  the  vegetative  cells  is  seen 
to  undergo  a  change.  It  loses  its  normal  homogeneous 
appearance  and  becomes  marked  by  granular,  refrac- 
tive points  of  irregular  shape  and  size.  These  eventu- 
ally coalesce,  leaving  the  remainder  of  the  cell  clear  and 
transparent.  When  this  coalescence  of  highly  refrac- 
tive particles  is  complete  the  spore  is  perfected.  In 
appearance,  the  spore  is  oval  or  round,  very  highly  re- 


44  BACTERIOLOGY. 

fractive,  and  of  a  glistening  appearance.  It  is  easily 
differentiated  from  the  remainder  of  the  cell,  which  now 
consists  only  of  a  cell-membrane  and  a  transparent, 
clear  fluid  which  surrounds  the  spore.  Eventually 
both  the  cell-membrane  and  its  fluid  contents  disappear, 
leaving  the  oval  spore  free. 

The  spore,  when  perfectly  developed,  is  highly  glis- 
tening, oval  in  contour,  and  has  the  appearance  of  being 
surrounded  by  a  dark,  sharply  defined  border.  It  pos- 
sesses no  motion  other  than  the  mechanical  tremor  com- 
mon to  all  insoluble  microscopic  particles  suspended  in 
fluids,  and  it  remains  quiescent  until  there  appear  con- 
ditions favorable  to  its  subsequent  development  into  the 
vegetative  form  from  which  it  originated.  Occasionally 
the  membrane  of  the  vegetative  cell  in  which  the  spore 
is  formed  does  not  disappear  from  around  it,  and  the 
spore  may  then  be  seen  lying  in  a  very  delicate  tubular 
envelope.  Now  and  then,  remnants  of  the  envelope  may 
be  noticed  adhering  to  the  spore  which  has  not  yet  be- 
come completely  free. 

When  stained,  the  spore-containing  cells  do  not  take 
up  the  dyes  in  a  homogeneous  way.  By  the  ordinary 
methods  the  spores  do  not  stain,  so  that  they  appear  in 
the  stained  cells  as  pale,  transparent,  oval  bodies,  sur- 
rounded by  the  remainder  of  the  cell,  which  has  taken 
up  the  staining. 

A  single  cell  produces  but  one  spore.  This  may  be 
located  either  at  an  extremity  or  in  the  centre  of  the  cell. 
(Fig.  4.) 

Occasionally  spore-formation  is  accompanied  by  an 
enlargement  of  the  cell  at  the  point  at  which  the  process 
is  in  progress.  As  a  result,  the  outline  of  the  cell  loses 
its  regular  rod  shape  and  becomes  that  of  a  club,  a 


MOTILITY.  45 

drum-stick,  or  a  lozenge,  depending  upon  whether  the 
location  of  the  spore  is  to  be  at  the  pole  or  in  the  centre 
of  the  cell.  (See  Fig.  4,  e  and  d.) 

In  addition  to  the  property  of  spore-formation  there 
is  another  striking  difference  between  the  rod-shaped 
organisms,  namely,  the  property  of  motility  which  many 
of  them  are  seen  to  possess.  This  power  of  motion  is 
due  to  the  possession  by  the  motile  bacilli  of  very  deli- 
cate, hair-like  appendages  or  flagella,  by  the  lashing 
motions  of  which  the  rods  possessing  them  are  propelled 
through  the  fluid.  In  some  cases  the  flagella  are  located 

FIG.  7. 


a  6  c 

a,  spiral  forms  with  a  flagellum  at  only  one  end ;  6,  bacillus  of  typhoid 
fever  with  flagella  given  off  from  all  sides ;  c,  large  spirals  from  stagnant 
water  with  wisps  of  flagella  at  their  ends  (spirillum  undula). 

at  but  one  end  of  a  bacillus,  either  singly  or  in  a 
bunch ;  again,  they  may  be  seen  at  both  poles,  and  in 
some  cases,  especially  with  the  bacillus  of  typhoid  fever, 
they  are  given  off  from  the  whole  surface  of  the  rod. 
(See  Fig.  7.)  In  a  few  instances  similar  locomotive 
organs  have  been  detected  on  spherical  bacteria,  i.  e., 
motile  micrococci  have  been  observed. 

For  a  long  time  the  motility  of  bacteria  was  only 
supposed  to  be  due  to  the  possession  of  some  such  form 
of  locomotive  apparatus  because  similar  appendages  had 
been  seen  in  certain  of  the  large,  motile  spirilla  found  in 


46  BACTERIOLOGY. 

stagDant  water,  and  it  was  not  until  recently  that  the 
accuracy  of  this  supposition  was  actually  demonstrated. 
By  a  special  method  of  staining,  Loeffler  has  been  able, 
in  a  number  of  cases,  to  render  visible  these  hair-like 
appendages.  His  method  consists  in  the  employment 
of  a  mordant,  by  the  aid  of  which  the  flagella  are  caused 
to  retain  the  staining,  and  thus  become  visible.  Loeffler's 
method  of  staining  will  be  found  in  the  chapter  devoted 
to  this  part  of  the  technique. 


CHAPTER   III. 


Principles  of  sterilization  by  heat— Methods  employed  —  Discontinued 
sterilization— Sterilization  under  pressure— Apparatus  employed— Chemical 
disinfection  and  sterilization. 


MOST  important  for  the  proper  performance  of  bac- 
teriological manipulations  is  an  acquaintance  with  the 
principles  underlying  the  methods  of  sterilization  and 
disinfection,  and  a  familiarity  with  the  approved  meth- 
ods of  applying  these  principles  in  practice. 

In  many  laboratories  it  is  customary  to  employ  the 
term  sterilization  for  the  destruction  of  bacteria  by 
heat,  and  the  term  disinfection  for  the  accomplishment 
of  the  same  end  through  the  use  of  chemical  agents. 
This  distinction  in  the  use  of  the  terms  is  not  strictly 
correct,  as  we  shall  endeavor  to  explain. 

The  laboratory  application  of  the  word  sterilization  for 
the  destruction  of  bacteria  by  high  temperatures  proba- 
bly arose  from  the  circumstance  that  culture  media,  and 
certain  other  articles  that  it  is  desirable  to  render  abso- 
lutely free  from  bacterial  life,  are  not  treated  by  chemical 
agents  for  this  purpose,  but  are  exposed  to  the  influ- 
ence of  heat  in  various  forms  of  apparatus  known  as 
sterilizers;  and  the  process  is,  therefore,  known  as 
sterilization.  On  the  other  hand,  cultures  no  longer 
useful,  bits  of  infected  tissue,  and  apparatus  generally, 
that  it  is  desirable  to  render  free  from  danger  are 
commonly  subjected  for  a  time  to  the  action  of  chemical 
compounds  possessing  germicidal  properties,  i.  e.,  to  the 


48  BACTERIOLOGY. 

action  of  disinfectants;  and  the  process  is,  therefore, 
known  as  disinfection,  though  the  same  end  can  also 
be  reached  by  the  application  of  heat  to  these  articles. 
Strictly  speaking,  sterilization  implies  the  complete  de- 
struction of  the  vitality  of  all  micro-organisms  that 
may  be  present  in  or  upon  the  substance  to  be  steril- 
ized, and  can  be  accomplished  by  the  proper  application 
of  both  thermal  and  chemical  agents ;  while  disinfec- 
tion, though  it  may,  need  not,  of  necessity,  insure  the 
destruction  of  all  living  forms  that  are  present,  but  only 
of  those  possessing  the  power  of  infecting;  it  may  or  may 
not,  therefore,  be  complete  in  the  sense  of  sterilization. 
From  this  we  see  it  is  possible  to  accomplish  both  steril- 
ization and  disinfection  as  well  by  chemical  as  by  ther- 
mal means. 

In  practice  the  employment  of  these  means  is  gov- 
erned by  circumstances.  In  the  laboratory  it  is  essen- 
tial that  all  culture  media  with  which  the  work  is  to  be 
conducted  should  be  free  from  living  bacteria  or  their 
spores — they  must  be  sterile — and  it  is  equally  impor- 
tant that  their  original  chemical  composition  should  re- 
main unchanged.  It  is  evident,  therefore,  that  steriliza- 
tion of  these  substances  by  means  of  chemicals  is  out  of 
the  question,  for,  while  the  media  could  be  thus  steril- 
ized, it  would  be  necessary,  in  order  to  accomplish  this, 
to  add  to  them  substances  capable  not  only  of  destroying 
all  micro-organisms  present,  but  whose  presence  would 
at  the  same  time  prevent  the  growth  of  bacteria  that  are  to 
be  subsequently  cultivated  in  these  media — that  is  to  say, 
after  performing  their  sterilizing  or  germicidal  function, 
the  chemical  disinfectants  would,  by  their  further  pres- 
ence, exhibit  their  antiseptic  properties  and  thus  render 
the  material  useless  as  a  culture  medium.  Exceptions 


STERILIZATION  BY  HEAT.  49 

to  this  are  seen,  however,  in  the  case  of  certain  volatile 
substances  possessing  disinfectant  powers,  chloroform  and 
ether,  for  instance ;  these  substances,  after  performing 
their  germicidal  activities,  may  be  driven  off  by  gentle 
heat,  leaving  the  media  quite  suitable  for  purposes  of 
cultivation.  They  are  not,  however,  in  general  use  in 
this  capacity. 

The  circumstances  under  which  chemical  sterilization 
or  disinfection  is  practised  in  the  laboratory  are  ordi- 
narily, either  those  in  which  it  is  desirable  to  render  ma- 
terials free  from  danger  that  are  not  affected  by  the 
chemical  action  of  the  agents  used,  such  as  glass  appa- 
ratus, etc.,  or  where  destructive  changes  in  the  compo- 
sition of  the  substances  to  be  treated,  as  in  the  case  of 
old  cultures,  infected  tissues,  etc.,  are  a  matter  of  no 
consequence.  On  the  other  hand,  for  the  sterilization 
of  all  materials  to  be  used  as  culture  media,  heat  only  is 
employed. 

The  two  processes  will  be  explained  in  this  chapter, 
beginning  with 

STERILIZATION   BY    HEAT. 

Sterilization  by  means  of  high  temperature  is  accom- 
plished in  several  ways,  viz.,  by  subjecting  the  sub- 
stance, to  be  treated  to  a  high  temperature  in  a  properly 
constructed  oven,  this  is  known  as  dry  sterilization ;  by 
subjecting  them  to  the  action  of  streaming  or  live  steam 
at  the  temperature  of  100°  C. ;  and  by  subjecting  them 
to  the  action  of  steam  under  pressure,  under  which 
circumstances  the  temperature  to  which  they  are  ex- 
posed becomes  more  and  more  elevated  as  the  pressure 
increases. 


50  BACTERIOLOGY. 

Experiments  have  taught  us  that  the  process  of  steril- 
ization by  dry  heat  has  a  relatively  limited  application 
because  of  its  many  disadvantages.  For  successful 
sterilization  by  the  method  of  dry  heat,  not  only  is  a 
relatively  high  temperature  essential,  but  the  substances 
under  treatment  must  be  exposed  to  this  temperature 
for  a  comparatively  long  time.  Its  penetration  into 
materials  which  are  to  be  sterilized  is,  moreover,  much 
less  energetic  than  that  of  steam.  Many  substances 
of  vegetable  and  animal  origin  are  rendered  useless 
by  subjection  to  the  dry  method  of  sterilization.  For 
these  reasons  there  are  comparatively  few  materials  that 
can  be  sterilized  in  this  way  without  seriously  impair- 
ing their  further  usefulness. 

Successful  sterilization  by  dry  heat  cannot  usually  be 
accomplished  at  a  temperature  lower  than  150°  C.,  and 
to  this  degree  of  heat  the  objects  should  be  subjected  for 
not  less  than  one  hour.  For  the  sterilization,  therefore, 
of  the  organic  materials  of  which  the  media  employed 
in  bacteriological  work  are  composed,  and  of  domestic 
articles,  such  as  cotton,  woollen,  wooden,  and  leather 
articles,  this  method  is  entirely  unsuitable.  In  bac- 
teriological work  its  application  is  limited  to  the  ster- 
ilization of  glassware  principally — such,  for  example, 
as  flasks,  plates,  small  dishes,  test-tubes,  pipettes — and 
such  metal  instruments  as  are  not  seriously  injured  by 
the  high  temperature. 

Sterilization  by  moist  heat — steam — offers  conditions 
much  more  favorable.  The  penetrating  action  of  the 
steam  is  not  only  more  energetic,  but  the  temperature 
at  which  sterilization  is  ordinarily  accomplished  is,  as  a 
rule,  not  destructive  to  the  objects  under  treatment.  This 
is  conspicuously  seen  in  the  work  of  the  laboratory ;  the 


UNIVERSITY 

OF 


STERILIZATION  BY  HEAT.  51 

culture  media,  composed  in  the  main  of  decomposable 
organic  materials  that  would  be  rendered  entirely  worth- 
less if  exposed  to  the  dry  method  of  sterilization,  sustain 
no  injury  whatever  when  intelligently  subjected  to  an 
equally  effective  sterilization  with  steam.  The  same 
may  be  said  of  cotton  and  woollen  fabrics,  bedding, 
clothing,  etc. 

Aside  from  the  relations  of  the  two  methods  to  the 
materials  to  be  sterilized,  their  action  toward  the  organ- 
isms to  be  destroyed  is  quite  different.  The  penetrating 
action  of  the  steam  renders  it  by  far  the  more  efficient 
agent  of  the  two.  The  spores  of  several  organisms 
which  are  killed  by  an  exposure  of  but  a  few  moments 
to  the  action  of  steam,  resist  the  destructive  action  of 
dry  heat  at  a  higher  temperature  for  a  much  greater 
length  of  time. 

These  differences  will  be  strikingly  brought  out  in  the 
experimental  work  on  this  subject.  For  our  purposes 
it  is  necessary  to  remember  that  the  two  methods  have 
the  following  applications : 

The  dry  method,  at  a  temperature  of  150°-180°  0., 
for  one  hour,  is  employed  for  the  sterilization  of  glass- 
ware :  flasks,  test-tubes,  culture  dishes,  pipettes,  plates, 
etc. 

The  sterilization  by  steam  is  practised  with  all  culture 
media,  whether  fluid  or  solid.  Bouillon,  milk,  gelatin, 
agar-agar,  potato,  etc.,  are  under  no  circumstances  to  be 
subjected  to  dry  heat. 

The  way  in  which  heat  is  employed  in  processes  of 
sterilization  varies  with  circumstances.  In  its  employ- 
ment as  dry  heat  its  application  is  always  continuous — 
i.  e.,  the  objects  to  be  sterilized  are  simply  exposed  to 
the  proper  temperature  for  the  length  of  time  necessary 


52  BACTERIOLOGY. 

to  destroy  all  living  organisms  which  may  be  upon  them. 
With  the  use  of  steam,  on  the  other  hand,  the  objects  to 
be  sterilized  are  frequently  of  such  a  nature  that  a 
prolonged  application  of  heat  might  materially  injure 
them.  For  this  and  other  reasons  steam  is  usually  ap- 
plied intermittently  and  for  short  periods  of  time.  The 
principles  involved  in  this  method  of  sterilization  depend 
upon  differences  of  resistance  toward  heat  which  the 
organisms  to  be  destroyed  are  seen  to  possess  at  different 
stages  of  their  development.  During  the  life  cycle  of 
many  of  the  bacilli  there  is  a  time  in  which  the  resist- 
ance of  the  organism  toward  the  action  of  both  chemical 
and  thermal  agents  is  much  higher  than  at  other  stages 
of  their  development.  This  increased  power  of  resistance 
appears  when  these  organisms  are  in  the  spore  or  resting 
stage,  to  which  reference  has  already  been  made.  When 
in  the  vegetative  or  growing  stage,  most  bacteria  are 
killed  in  a  short  time  by  a  relatively  low  temperature, 
whereas,  when  conditions  have  arisen  which  favor  the 
production  of  spores,  the  spores  are  seen  to  be  capable  of 
resisting  very  much  higher  temperatures  for  an  appre- 
ciably longer  time,  indeed  spores  of  certain  bacilli  have 
been  encountered  that  retain  their  power  of  germinating 
after  an  exposure  of  from  five  to  six  hours  to  the  tem- 
perature of  boiling  water.  Such  powers  of  resistance 
have  never  been  observed  in  the  vegetating  stage  of 
development.  These  differences  in  resistance  toward 
heat  which  the  spore-forming  organisms  possess  at 
their  different  stages  of  development  are  taken  advan- 
tage of  in  that  process  of  sterilization  by  steam  known 
as  the  fractional  or  intermittent  method,  and  form  the 
principle  on  which  the  method  is  based. 

As  the  culture  media  to  be  sterilized  are  dependent 


STERILIZA  TION  B  Y  HE  A  T.  53 

for  their  value  upon  the  presence  of  more  or  less 
unstable  organic  compounds,  the  object  aimed  at  in  this 
method  is  to  destroy  the  organisms  in  the  shortest 
time  and  with  the  least  amount  of  heat.  It  is  accom- 
plished by  subjecting  them  to  the  elevated  temperature  at 
a  time  when  the  bacteria  are  in  the  vegetating  or  growing 
stage — i.  e.j  the  stage  at  which  they  are  most  susceptible 
to  detrimental  agencies.  In  order  to  accomplish  this  it 
is  necessary  that  there  should  exist  conditions  of  tem- 
perature, nutrition,  and  moisture  which  favor  the  vege- 
tation of  the  bacilli  and  the  germination  of  any  spores 
that  may  be  present.  When,  as  in  freshly  prepared 
nutrient  media,  these  surroundings  are  found,  the  spore- 
forming  organisms  are  not  only  less  likely  to  enter  the 
spore  stage  than  when  their  environments  are  less  favor- 
able to  their  vegetation,  but  spores  which  may  already 
exist  develop  very  quickly  into  mature  cells, 

It  is  plain,  then,  that  with  the  first  application  of  the 
steam  to  the  substance  to  be  sterilized  the  mature  vege- 
tative forms  of  these  organisms  are  destroyed,  while  cer- 
tain spores  that  might  have  been  present  resist  this 
treatment,  providing  the  sterilization  has  not  been  con- 
tinued for  too  long  a  time.  If  now  the  sterilization  is 
discontinued,  and  the  material  which  presents  conditions 
favorable  to  the  germination  of  the  spores  is  allowed  to 
stand  for  a  time,  usually  for  about  twenty-four  hours, 
at  a  temperature  of  from  20°-30°  C.,  those  spores  which 
resisted  the  action  of  the  steam  will,  in  the  course  of  this 
interval,  germinate  into  the  less  resistant  vegetative  cells. 
A  second  short  exposure  to  the  steam  kills  these  forms 
in  turn,  and  by  a  repetition  of  this  process  all  organisms 
which  were  present  may  be  destroyed  without  the  appli- 
cation of  the  steam  having  been  of  long  duration  at  any 


54  BACTERIOLOGY. 

time.  It  should  be  remembered  that  while  spores  which 
may  be  present  are  not  directly  killed  by  the  exposure 
to  heat  that  they  experience  in  the  intermittent  method 
of  sterilization,  still  their  power  of  germination  is  some- 
what inhibited  by  this  treatment.  In  this  method, 
therefore,  if  the  temperature  of  100°  C.  be  employed  for 
too  long  a  time,  it  is  possible  to  so  retard  the  germina- 
tion of  the  spores  as  to  render  it  impossible  for  them  to 
develop  into  the  vegetating  stage  during  the  interval 
between  the  heatings.  By  excessively  long  exposures 
to  high  temperature,  but  not  long  enough  to  destroy  the 
spores  directly,  the  object  aimed  at  in  the  method  may 
be  defeated,  and  in  the  end  the  substance  undergoing 
sterilization  be  found  to  still  contain  living  bacteria.  In 
this  process  the  plan  that  has  given  most  satisfactory 
results  is  to  subject  the  materials  to  be  sterilized  to  the 
action  of  steam,  under  the  ordinary  conditions  of  atmos- 
pheric pressure,  for  fifteen  minutes  on  each  of  three 
successive  days,  and  during  the  intervals  to  retain  them 
at  a  temperature  of  about  25°-30°  C.  At  the  end  of 
this  time  all  living  organisms  which  were  present  will 
have  been  destroyed,  and,  unless  opportunity  is  given 
for  the  access  of  new  organisms  from  without,  the  sub- 
stances thus  treated  remain  sterile. 

As  an  exception  to  this,  one  occasionally  encounters 
certain  species  of  spore-forming  bacteria  that  are  not 
readily  destroyed  by  this  mode  of  treatment.  They  are, 
presumably,  of  the  group  of  so-called  "soil  organisms/7 
and  represent  the  forms  most  resistant  to  the  influence 
of  heat.  We  are  not  as  yet  sufficiently  familiar  with 
all  their  peculiarities  to  warrant  our  speaking  with  cer- 
tainty as  to  a  means  of  sterilizing  media  in  which  they 
are  present.  It  does  not  seem  unlikely  that  they  are  of 


STERILIZATION  BY  HEAT.  55 

the  thermophilic  (possibly  facultative  thermophilic)  va- 
riety, and  show  little  tendency  to  develop  into  the  vegeta- 
tive stage  between  the  heatings,  germinating  perhaps  so 
slowly  at  the  temperature  under  which  they  find  them- 
selves as  not  to  completely  leave  the  spore  stage  before 
another  exposure  to  the  steam,  but  manifesting  after  a 
time  properties  of  life  in  the  media  that  is  thought  to  be 
sterile  arid  which  has  been  placed  aside  for  subsequent  use. 
This  is  a  mere  hypothesis,  however,  and  is  as  yet  entirely 
.wanting  in  experimental  proof. 

Fortunately,  these  undesirable  experiences  are  rare, 
but  that  they  do  occur,  and  result  in  no  small  degree  of 
annoyance,  is  an  experience  that  has  probably  been  had 
by  most  bacteriologists. 

It  must  be  borne  in  mind  that  this  method  of  sterili- 
zation is  only  applicable  in  those  cases  which  present 
conditions  favorable  to  the  germination  of  the  spores 
into  mature  vegetative  cells.  Dry  substances  or  organic 
materials  in  which  decomposition  is  far  advanced,  where 
conditions  of  nutrition  favorable  to  the  germination  of 
spores  are  not  present,  cannot  be  successfully  sterilized 
by  the  intermittent  method. 

The  process  of  fractional  sterilization  at  low  tempera- 
tures is  based  upon  exactly  the  same  principle,  but  dif- 
fers from  the  foregoing  in  the  method  by  which  it  is 
practised  in  two  respects,  viz.,  it  requires  a  greater  num- 
ber of  exposures  for  its  accomplishment,  and  the  tem- 
perature at  which  it  is  conducted  is  not  raised  above 
68°-70°  C.  It  is  employed  for  the  sterilization  of  easily 
decomposable  materials,  which  would  be  rendered  use- 
less by  the  temperature  of  steam,  but  which  remain 
intact  at  the  temperature  employed,  and  for  certain 
albuminous  culture  media  that  it  is  desirable  to  retain 


56  BACTERIOLOGY. 

in  a  fluid  condition  during  sterilization,  but  which  would 
be  coagulated  if  exposed  to  high  temperatures.  This 
process  requires  that  the  material  to  be  sterilized  should 
be  subjected  to  a  temperature  of  68°-70°  C.  for  one 
hour  on  each  of  six  successive  days,  the  interval  of 
twenty-four  hours  between  the  exposures  admitting  of 
the  germination  of  spores  into  mature  cells.  During 
this  interval  the  substances  under  treatment  are  kept 
at  about  25°-30°  C.  The  temperature  employed  in  this 
process  suffices  to  destroy  the  vitality  of  almost  all 
organisms  in  the  vegetative  stage  in  about  one  hour. 
Until  recently  blood-serum  was  always  sterilized  by  the 
intermittent  method  at  low  temperature. 

Sterilization  by  steam  is  also  practised  by  what  may 
be  called  the  direct  method.  That  is  to  say,  both  the 
mature  organisms  and  the  spores  which  may  be  present 
in  the  material  to  be  sterilized  are  destroyed  by  a  single 
exposure  to  the  steam.  In  this  method  steam  at  its 
ordinary  temperature  and  pressure — live  steam  or  stream- 
ing steam  as  it  is  called — is  employed  just  as  in  the  first 
method  described,  but  it  is  allowed  to  act  for  a  much 
longer  time,  usually  not  less  than  one  hour ;  or,  steam 
under  pressure,  and  consequently  of  a  higher  tempera- 
ture, is  now  frequently  employed.  By  the  latter  pro- 
cedure a  single  exposure  of  fifteen  minutes  is  sufficient 
for  the  destruction  of  practically  all  bacilli  and  their 
spores,  providing  the  pressure  of  the  steam  is  not  less 
than  one  atmosphere  over  and  above  that  of  normal — 
this  is  approximately  equivalent  to  a  temperature  of 
122°  C.  to  which  the  organisms  are  exposed. 

The  objection  that  has  been  urged  to  both  of  these 
methods,  particularly  that  in  which  steam  under  press- 
ure is  employed,  is  that  the  constitution  of  the  media  is 


STERILIZA  TION  B  Y  HE  A  T.  57 

altered.  Gelatiii  is  said  to  become  cloudy  and  lose  the 
property  of  solidifying ;  in  bouillon  and  agar-agar  fine 
precipitates  are  thought  to  occur,  and  some  think  the 
reaction  undergoes  a  change.  In  the  experience  of  those 
who  have  used  steam  under  pressure,  not  exceeding  one 
or  one  and  one-half  atmospheres  for  a  few  minutes,  ten 
to  fifteen,  these  obstacles  have  rarely  been  encountered. 
There  is  one  point  to  be  borne  in  mind,  however,  in 
using  steam  under  pressure,  viz.,  it  is  not  possible  to 
regulate  the  time  of  exposure  to  the  same  degree  of  nicety 
as  where  ordinary  live  steam  is  used.  The  reason  for 
this  is  that  if  the  apparatus  be  opened  while  the  steam 
that  is  within  it  is  under  pressure,  the  escape  of  steam 
will  be  so  rapid  that  all  fluids  within  the  chambers,  thus 
suddenly  relieved  of  pressure,  will  begin  to  boil  violently, 
and,  as  a  rule,  will  boil  quite  out  of  the  tubes,  flasks,  etc., 
containing  them.  For  this  reason  the  apparatus  must 
be  kept  closed  until  cool,  or  until  the  steam  gauge  indi- 
cates that  pressure  no  longer  exists  within  the  chambers, 
and  even  then  it  should  be  opened  very  cautiously.  It 
is  patent  from  this  that  the  time  and  temperature  of  ex- 
posure of  articles  sterilized  by  this  process  cannot  usually 
be  controlled  with  accuracy.  It  requires  some  time  to 
reach  a  given  pressure  after  the  apparatus  is  closed,  and 
it  abo  requires  time  for  cooling  down  after  the  desired 
exposure  to  such  pressure  before  the  apparatus  can  be 
opened. 

It  is  manifest  that  during  these  three  periods,  viz., 
(a)  reaching  the  pressure  desired,  (6)  time  during  which 
the  pressure  is  maintained,  and  (c)  time  for  fall  of  press- 
ure before  the  chamber  can  be  opened,  it  is  difficult  to 
say  certainly  to  what  temperature  and  pressure  the  arti- 
cles in  the  apparatus  have,  on  the  whole,  been  subjected. 


58  BACTERIOLOGY. 

Clearly,  if  the  desired  pressure  and  temperature  have 
been  maintained  for  ten  minutes,  one  cannot  say  that 
this  is  all  the  heat  to  which  the  articles  have  been  sub- 
jected during  their  stay  in  the  chamber.  In  this  light, 
while  steam  under  pressure  may  answer  very  well  for 
routine  sterilization,  still  it  presents  insurmountable 
obstacles  to  its  use  in  finer  experiments  where  time  ex- 
posure to  definite  temperature  is  of  importance. 

For  most  of  the  media  which  are  employed  the  discon- 
tinued method  at  the  temperature  of  streaming  steam 
gives  the  most  satisfactory  results. 

For  sterilization  by  live  steam  the  apparatus  com- 
monly employed  has,  until  recently,  been  the  cylindrical 
boiler  recommended  by  Koch.  (See  Fig.  8.) 

Its  construction  is  very  simple.  It  consists  of  a 
copper  cylinder,  the  lower  fifth  of  which  is  somewhat 
larger  in  diameter  than  the  remaining  four-fifths,  and 
acts  as  a  reservoir  for  the  water  from  which  the  steam 
is  to  be  generated.  Covering  this  section  of  the  cylinder 
is  a  wire  rack  or  grating  through  which  the  steam 
passes,  and  which  serves  as  a  bottom  upon  which  the 
objects  to  be  sterilized  rest.  Above  this,  comprising 
the  remaining  four-fifths  of  the  cylinder,  is  the  cham- 
ber for  the  reception  of  the  materials  over  and  through 
which  the  steam  is  to  pass.  The  cylinder  is  closed  by 
a  snugly-fitting  cover  through  which  are  usually  two 
perforations  into  which  a  thermometer  and  a  manometer 
may  be  inserted.  The  whole  of  the  outer  surface  of 
the  apparatus  is  encased  in  a  non-conducting  mantle  of 
asbestos  or  felt. 

The  water  is  heated  by  a  gas-flame  placed  in  an  en- 
closed chamber,  upon  which  the  apparatus  rests,  which 
serves  to  diminish  the  loss  of  heat  and  deflection  of  the 


STERILIZATION  BY  HEAT.  59 

flame  through  the  action  of  draughts.  The  apparatus 
is  simple  in  construction,  and  the  only  point  which  is  to 
be  observed  while  using  it  is  the  level  of  the  water  in 


Steam  sterilizer,  pattern  of  Koch. 

the  reservoir.  On  the  reservoir  is  a  water-gauge  which 
indicates  at  all  times  the  amount  of  water  in  the  appa- 
ratus. The  amount  of  water  should  never  be  too  small 
to  be  indicated  by  the  gauge,  otherwise  there  is  danger 
of  the  reservoir  becoming  dry  and  the  bottom  of  the 
apparatus  being  destroyed  by  the  direct  action  of  the 
flame. 

A  sterilizer  that  has  come  into  very  general  use  in 
bacteriological  laboratories  is  one  originally  intended 
for  use  in  the  kitchen.  It  is  the  so-called  "Arnold 


60  BACTERIOLOGY. 

Steam  Sterilizer."     It  is  very  ingenious  in  its  construc- 
tion as  well  as  economical  in  its  employment. 

The  difference  between  this  apparatus  and  that  just 
described  is  that  it  provides  for  the  condensation  of  the 
steam  after  its  escape  from  the  sterilizing  chamber,  and 
returns  the  water  of  condensation  automatically  to  the 
reservoir,  so  that  in  practice  the  apparatus  requires  but 

FIG.  9. 


Arnold  steam  sterilizer. 


little  attention,  as  with  moderate  care  there  is  no  fear  of 
the  water  in  the  reservoir  becoming  exhausted  and  the 
consequent  destruction  of  the  sterilizer. 

Fig.  9  shows  a  section  through  this  apparatus. 

STERILIZATION    UNDER   PRESSURE. 

For  sterilization  by  steam  under  pressure  several  spe- 
cial forms  of  apparatus  exist.     The  principles  involved 


STERILIZATION  UNDER  PRESSURE. 


61 


in  them  all  are,  however,  the  same.  They  provide  for 
the  generation  of  steam  in  a  chamber  from  which  it  can- 
not escape  when  the  apparatus  is  closed.  Upon  the  cover 


FIG.  10. 


Autoclave  or  digester  for  sterilizing  by  steam  under  pressure. 

of  this  chamber  is  a  safety-valve,  which  can  be  regulated 
so  that  any  degree  of  pressure  (and  coincidently  of  tem- 
perature) that  is  desirable  can  be  maintained  within  the 
sterilizing  chamber.  These  sterilizers  are  known  as 
"  digesters  "  and  as  "  autoclaves."  Their  construction 
can  best  be  understood  by  reference  to  Fig.  10. 


62 


BACTERIOLOGY. 


STERILIZATION   BY   HOT   AIR. 

The  hot-air  sterilizers  used  in  laboratories  are  simply 
double-walled  boxes  of  Russian  or  Swedish  iron  (Fig. 
11),  having  a  double- walled  door,  which  closes  tightly, 
and  a  heavy  copper  bottom.  They  are  arranged  with 
ventilating  openings  for  the  escape  of  the  contained  air 
and  the  entrance  of  the  heated  air.  The  flame,  usually 
from  a  rose  burner  (Fig.  12),  is  applied  directly  to  the 
bottom.  The  heat  circulates  from  the  lower  surface 
around  about  the  apparatus  through  the  space  between 
its  walls. 


FIG.  11. 


PIG.  12. 


The  construction  of  the  copper  bottom  of  the  appa- 
ratus upon  which  the  flame  impinges  is  designed  to 
prevent  the  direct  action  of  the  flame  upon  the  sheet- 


CHEMICAL  STERILIZA  TION  AND  DISINFECTION.       63 

iron  bottom  of  the  chamber.  It  consists  of  several 
copper  plates  placed  one  above  the  other,  but  with  a 
space  of  about  4  to  5  mm.  between  the  plates.  These 
copper  bottoms  after  a  time  become  burned  out,  and 
unless  they  are  replaced  the  apparatus  is  useless.  The 
older  forms  of  hot-air  sterilizers  are  so  constructed  that 
their  repair  is  a  matter  involving  some  time  and  ex- 
pense. To  meet  this  objection  I  have  had  constructed  a 
sterilizer  in  all  respects  similar  to  the  old  form  except  in 
the  arrangement  of  this  copper  bottom.  This  is  made 
in  such  a  way  that  it  can  be  easily  removed,  so  that  by 
keeping  several  sets  of  copper  plates  on  hand  a  new  one 
can  readily  be  inserted  when  the  old  one  is  burned  out. 

In  the  employment  of  the  hot-air  sterilizer  care  should 
always  be  given  to  the  condition  of  the  copper  bottom  ; 
for  the  direct  application  of  the  heat  to  the  sheet-iron 
plate  upon  which  the  substances  to  be  sterilized  stand, 
results  not  only  in  destruction  of  the  apparatus,  but  fre- 
quently in  destruction  of  the  substances  undergoing  ster- 
ilization. 

Since  the  temperature  at  which  this  form  of  steriliza- 
tion is  usually  accomplished  is  high,  from  150°  to  180° 
C.,  it  is  well  to  have  the  apparatus  encased  in  asbestos 
boards,  to  diminish  the  radiation  of  heat  from  its  sur- 
faces. This  not  only  confines  the  heat  to  the  apparatus, 
but  guards  against  the  destructive  action  of  the  radiated 
heat  on  woodwork,  furniture,  etc.,  that  may  be  in  the 
neighborhood. 

CHEMFCAL   STERILIZATION    AND   DISINFECTION. 

As  has  already  been  stated,  it  is  possible  by  means 
of  certain  chemical  substances  to  destroy  all  bacteria 


64  BACTERIOLOGY. 

and  their  spores,  that  may  be  within  or  upon  various 
materials  and  objects,  i.  e.,  to  sterilize  them ;  and  it  is 
also  possible  by  the  same  means  to  rob  infected  objects 
of  their  dangerous  infective  properties  without  at  the 
same  time  sterilizing  them,  i.  e.,  to  disinfect  them. 
This  latter  process  depends  upon  the  fact  that  the 
vitality  of  many  of  the  less  resistant  pathogenic  organ- 
isms is  easily  destroyed  by  an  exposure  to  particular 
chemical  substances,  while  a  similar  exposure  may  be 
without  effect  upon  the  more  resistant  saprophytes  and 
their  spores  that  are  present. 

In  general  the  use  of  chemicals  for  sterilization  is  not 
to  be  considered  in  connection  with  substances  that  are 
to  be  employed  as  culture  media,  and  their  employment 
is  restricted  in  the  laboratory  to  materials  that  are  of  no 
further  value,  and  to  infected  articles  that  are  not  in- 
jured by  the  action  of  the  agents  used,  though  for  par- 
ticular purposes  such  volatile  germicides  as  chloroform 
and  ether  may  serve  as  exceptions  to  this.  (See  Pres- 
ervation of  Blood-serum  with  Chloroform,  page  90.) 
In  short,  they  are  mainly  of  value  in  rendering  infected 
waste  materials  free  from  danger.  For  the  successful 
performance  of  this  form  of  disinfection  there  is  one 
fundamental  rule  always  to  be  borne  in  mind,  viz.,  it  is 
absolutely  essential  to  success  that  the  disinfectant  used 
should  come  in  direct  contact  with  the  bacteria  to  be 
destroyed,  otherwise  there  is  no  disinfection. 

For  this  reason,  one  should  always  remember,  in 
selecting  the  disinfecting  agent,  the  nature  of  the  ma- 
terials containing  the  bacteria  upon  which  it  is  to  act, 
for  the  majority  of  disinfectants,  and  particularly  those 
of  an  inorganic  nature,  vary  in  the  degree  of  their 
potency  with  the  chemical  nature  of  the  mass  to  which 


CHEMICAL  STERILIZATION  AND  DISINFECTION.       65 

they  are  applied.  Often  the  materials  containing  the 
bacteria  to  be  destroyed  are  of  such  a  character  that 
they  combine  with  the  disinfecting  agent  to  form  insol- 
uble precipitates ;  these  so  interfere  with  the  penetration 
of  the  disinfectant  that  many  bacteria  may  escape  its 
destructive  action  entirely  and  no  disinfection  be  accom- 
plished, though  an  agent  might  have  been  employed 
that  would,  under  other  circumstances,  have  given 
entirely  satisfactory  results. 

In  the  destruction  of  bacteria  by  means  of  chemical 
substances  there  occurs,  most  probably,  a  definite  chemi- 
cal reaction ;  that  is  to  say,  the  characteristics  of  both 
the  bacteria  and  the  agent  employed  in  their  destruction 
are  lost  in  the  production  of  an  inert  third  body,  the 
result  of  their  combination.  It  is  impossible  to  say  with 
absolute  certainty,  as  yet,  that  this  is  the  case,  but  the 
evidence  that  is  rapidly  accruing  from  the  more  recent 
studies  upon  disinfectants  and  their  mode  of  action  points 
strongly  to  the  accuracy  of  this  belief.  This  reaction,  in 
which  the  typical  structures  of  both  bodies  concerned  is 
lost,  takes  place  between  the  agent  employed  for  disin- 
fection and  the  protoplasm  of  the  bacteria.  For  ex- 
ample, in  the  reaction  that  is  seen  to  take  place  between 
the  salts  of  mercury  and  albuminous  bodies  there  results 
a  third  compound,  which  has  neither  the  characteristics 
of  mercury  nor  of  albumin,  but  partakes  of  the  pecu- 
liarities of  both ;  it  is  a  combination  of  albumin  and 
mercury  known  by  the  indefinite  term  "albuminate  of 
mercury."  Some  such  reaction  as  this  occurs  when  the 
soluble  salts  of  mercury  are  brought  in  contact  with 
bacteria.  This  view  has  recently  been  strengthened  by 
the  experiments  of  Geppert,  in  which  the  reaction  was 
caused  to  take  place  between  the  spores  of  the  anthrax 


66  BACTERIOLOGY. 

bacillus  and  a  solution  of  mercuric  chloride,  the  result 
being  the  apparent  destruction  of  the  vitality  of  the 
spores  by  the  formation  of  this  third  compound.  In 
these  experiments  it  was  shown  that  though  this  com- 
bination had  taken  place,  still  it  did  not  of  necessity 
imply  the  complete  death  of  the  protoplasm  of  the 
spores,  for  if  by  proper  means  the  combination  of  mer- 
cury with  their  protoplasm  was  broken  up,  many  of 
the  spores  returned  from  their  condition  of  apparent 
death  to  that  of  life,  with  all  their  previous  disease- 
producing  and  cultural  peculiarities.  Geppert  employed 
a  solution  of  ammonium  sulphide  for  the  purpose  of  de- 
stroying the  combination  of  spore-protoplasm  and  mer- 
cury ;  the  mercury  was  precipitated  from  the  proto- 
plasm as  an  insoluble  sulphide,  and  the  protoplasm  of 
the  spores  returned  to  its  original  condition.  These  and 
other  somewhat  similar  experiments  have  given  an 
entirely  new  impulse  to  the  study  of  disinfectants,  and 
in  the  light  shed  by  them  many  of  our  previously 
formed  ideas  concerning  the  action  of  disinfecting 
agents  must  be  modified.  The  process  is  not  a  catalytic 
one — i.  e.,  occurring  simply  as  a  result  of  the  presence 
of  the  disinfecting  body  which  is  not  of  itself  destroyed 
in  its  process  of  destruction — but  is,  as  said,  a  definite 
chemical  reaction  which  takes  place  within  certain  more 
or  less  fixed  limits  ;  that  is  to  say,  with  a  given  amount 
of  the  disinfectant  employed,  just  so  much  work,  ex- 
pressed in  terms  of  disinfection — destruction  of  bacteria 
— can  be  accomplished. 

Another  point  in  favor  of  this  view  is  the  increased 
energy  of  the  reaction  with  elevation  of  temperature. 
Just  as  in  many  other  chemical  phenomena,  the  intensity 
of  the  reaction  becomes  greater  under  the  influence  of 


CHEMICAL  STERILIZATION  AND  DISINFECTION.       67 

heat,  so  in  the  process  of  disinfection  the  combination 
between  the  disinfectant  and  the  organisms  to  be  de- 
stroyed is  much  more  energetic  at  a  temperature  of  37° 
to  39°  C.  than  it  is  at  12°  to  15°  C. 

What  has  been  said  refers  more  particularly  to  the 
inorganic  salts  which  are  employed  for  this  purpose.  It 
is  probable  that  the  organic  bodies  which  possess  disin- 
fectant properties  owe  this  power  to  some  such  similar 
reaction,  though,  as  yet,  these  substances  have  not  been 
so  thoroughly  studied  in  this  relation. 

The  reaction  between  these  inorganic  salts  and  albu- 
minous bodies  is  not  selective ;  they  combine  in  most 
instances  with  any  or  all  protoplasmic  bodies  present. 
For  this  reason  the  results  of  the  practical  application 
of  many  of  the  commonly  employed  disinfectants  is 
a  matter  of  grave  doubt.  For  example,  the  disinfec- 
tion of  excreta,  sputum,  or  blood  containing  pathogenic 
organisms,  by  means  of  corrosive  sublimate,  is  a  proce- 
dure of  very  questionable  success.  The  amount  of  sub- 
limate employed  may  be  entirely  used  up  and  rendered 
inactive  as  a  disinfectant  by  the  ordinary  protoplasmic 
substances  present,  without  having  any  appreciable  ef- 
fect upon  the  bacteria  which  may  be  in  the  mass. 

These  remarks  are  introduced  in  order  to  guard 
against  the  implicit  confidence  so  often  placed  in  the 
disinfecting  value  of  corrosive  sublimate.  In  bacterio- 
logical laboratories,  where  there  is  constantly  more  or 
less  of  infectious  material,  it  is  the  custom,  with  few 
exceptions,  to  have  vessels  containing  solutions  of  cor- 
rosive sublimate  at  hand,  by  which  infectious  materials 
may  be  rendered  harmless.  The  value  of  this  pro- 
cedure, as  we  have  just  learned,  is  always  more  or  less 
questionable,  especially  in  those  cases  in  which  the  sub- 


68  BACTERIOLOGY. 

stance  to  be  disinfected  is  of  a  proteid  nature.  With 
the  introduction  of  such  substances  into  the  sublimate 
solution  the  mercury  is  quickly  precipitated  by  the 
albumin,  and  its  disinfecting  properties  may  be  entirely 
destroyed ;  we  may  in  a  very  short  time  have  little  else 
than  water  containing  a  precipitate  of  albumin  and  mer- 
cury, in  so  far  as  its  value  as  a  disinfectant  is  concerned. 

Though  the  other  inorganic  salts  have  not  been  so 
thoroughly  studied  in  this  connection,  it  is  nevertheless 
probable  that  the  same  precautions  should  be  taken  in 
their  employment  as  we  now  know  to  be  necessary  in 
the  use  of  the  salts  of  mercury. 

Where  it  is  desirable  to  use  chemical  disinfectants  in 
the  laboratory,  much  more  satisfactory  results  can 
usually  be  obtained  through  the  employment  of  carbolic 
acid  in  solution.  A  three  or  four  per  cent,  solution  of 
commercial  carbolic  acid  in  water  requires  a  somewhat 
longer  time  for  disinfection,  but  it  is,  at  the  same  time, 
open  to  fewer  objections  than  are  solutions  of  the  in- 
organic salts,  though  here,  too,  we  find  a  somewhat 
analogous  reaction  between  the  carbolic  acid  and  proteid 
matters.  Under  ordinary  circumstances  its  action  is 
complete  in  from  twenty  minutes  to  one-half  hour. 

In  the  laboratory  heat  is  the  surest  agent  to  employ. 
All  tissues  containing  infectious  organisms  should  be 
burned,  and  all  cloths,  test-tubes,  flasks,  and  dishes 
should  be  boiled  in  2  per  cent,  soda  (ordinary  washing 
soda)  solution  for  fifteen  to  twenty  minutes,  or  placed 
in  the  steam  sterilizer  for  half  an  hour. 

Intestinal  evacuations  may  best  be  disinfected  with 
boiling  water  or  with  milk  of  lime,  a  mixture  composed 
of  lime  in  solution  and  in  suspension,  ordinary  fluid 
"  white-wash."  This  should  be  thoroughly  mixed  with 


CHEMICAL  STERILIZA  TION  AND  DISINFECTION.       6  9 

the  evacuations  until  the  mass  reacts  distinctly  alkaline, 
and  should  remain  in  contact  with  the  infective  sub- 
stance for  one  or  two  hours.  If  boiling  water  be  used 
the  amount  should  be  about  double  the  volume  of  the 
mass  to  be  disinfected.  They  should  be  thoroughly 
mixed  and  allowed  to  stand,  covered,  until  cold. 

Sputum  in  which  tubercle  bacilli  are  present,  as  well 
as  the  vessel  containing  it,  must  be  boiled  in  2  per  cent, 
soda  solution  for  fifteen  minutes,  or  steamed  in  the 
sterilizer  for  at  least  half  an  hour. 

On  the  whole,  in  the  laboratory  we  should  as  yet 
rely  more  upon  the  destructive  properties  of  heat  than 
upon  those  of  chemical  agents. 

From  what  has  been  said,  the  absurdity  of  sprinkling 
about,  here  and  there,  a  little  carbolic  acid  or  in  placing 
about  apartments  in  which  infectious  diseases  are  in 
progress  little  vessels  of  carbolic  acid,  must  be  plain. 
The  disinfection  of  water-closets  and  cesspools  by  allow- 
ing now  and  then  a  few  cubic  centimetres  of  some  so- 
called  disinfectant  to  trickle  through  the  pipes  is  ridic- 
ulous. A  disinfectant  must  be  applied  to  the  bacteria, 
and  must  be  in  contact  with  them  for  a  long  enough  time 
to  insure  the  destruction  of  their  life. 

In  the  light  of  the  latest  experiments  upon  disinfec- 
tants, the  place  formerly  occupied  by  many  agents  in  the 
list  of  substances  employed  for  the  purpose,  will  most 
likely  be  changed  as  they  are  studied  more  closely. 

The  agents,  then,  which  will  prove  of  most  value  in 
the  laboratory  for  the  purpose  of  rendering  infectious 
materials  harmless  are :  Heat,  either  by  burning,  by 
steaming  for  from  half  an  hour  to  an  hour,  or  by  boil- 
ing in  a  2  per  cent,  sodium  carbonate  solution  for  fifteen 
minutes ;  3  to  4  per  cent,  solution  of  commercial  car- 

4* 


70  BACTERIOLOGY. 

bolic  acid ;  milk  of  lime,  and  a  solution  of  chlorinated 
lime  ("chloride  of  lime"),  containing  not  less  than  0.25 
per  cent,  of  free  chlorine.  The  chloride  of  lime  from 
which  such  a  solution  is  to  be  made  should  be  fresh  and 
of  good  quality.  Good  chloriuated  lime,  as  purchased 
in  the  shops,  should  contain  not  less  than  25  to  30  per 
cent,  of  available  chlorine.  The  materials  to  be  dis- 
infected in  either  of  the  lime  solutions  should  remain 
in  them  for  about  two  hours.  The  solutions  should  be 
freshly  prepared  when  needed,  as  they  rapidly  decom- 
pose upon  standing. 

Antiseptic.  An  antiseptic  is  a  body  which,  by  its 
presence,  prevents  the  growth  of  bacteria  without  of 
necessity  killing  them.  A  body  may  be  an  antiseptic 
without  possessing  disinfecting  properties  to  any  very 
high  degree,  but  a  disinfectant  is  always  an  antiseptic 
as  well. 


CHAPTEK    IV. 

Principles  involved  in  the  methods  of  isolation  of  bacteria  in  pure  culture 
by  the  plate  method  of  Koch— Materials  employed. 

As  was  stated  in  the  introductory  chapter,  the  isola- 
tion in  pure  cultures  of  the  different  species  of  bacteria 
from  mixtures  of  these  organisms  was  rendered  possible 
only  through  the  methods  suggested  by  Koch.  Since 
the  adoption  of  these  methods  they  have  undergone 
many  modifications,  but  the  original  principle  involved 
has  remained  unaltered.  The  observation  which  led  to 
their  development  was  a  very  simple  one,  and  one  that 
is  commonly  before  us.  Koch  noticed  that  on  solid  sub- 
stances, such,  for  example,  as  a  slice  of  potato  or  of 
bread,  which  had  been  exposed  for  a  time  to  the  air  and 
which  afforded  proper  nourishment  for  the  lower  organ- 
isms, there  developed  after  a  short  time  small  patches  of 
material  which  proved  to  be  colonies  of  bacteria.  Each 
of  these  colonies  on  closer  examination  showed  itself  to 
be,  as  a  rule,  composed  of  but  a  single  species.  There 
was  little  tendency  on  the  part  of  these  colonies  to  be- 
come confluent,  and  from  the  differences  in  their  naked- 
eye  appearances,  it  was  easy  to  see  that  they  were  mostly 
the  outgrowth  of  different  species  of  bacteria. 

The  question  that  then  presented  itself  was  :  If  from 
a  mixture  of  organisms  floating  in  the  air  it  is  possible 
in  this  way  to  obtain  in  pure  cultures  the  component 
individuals,  what  means  can  be  employed  for  obtain- 
ing the  same  results  at  will  from  mixtures  of  different 


72  BACTERIOLOGY. 

species  of  bacteria  when  found  together  under  other 
conditions  ? 

It  was  plain  that  the  organisms  were  to  be  distin- 
guished primarily,  the  one  from  the  other,  only  by  the 
structure  and  general  appearance  of  the  colonies  grow- 
ing from  them,  for  by  their  morphology  alone  this  is 
impossible. 

What  means  could  be  devised,  then,  for  separating  the 
individual  members  of  a  mixture  in  such  a  way  that 
they  would  remain  in  a  fixed  position,  and  be  so  widely 
separated,  the  one  from  the  other,  as  not  to  interfere  with 
the  production  of  colonies  of  characteristic  appearance, 
which  would,  under  the  proper  conditions,  develop  from 
each  individual  cell  ? 

If  one  takes  in  the  hand  a  mixture  of  barley,  rye, 
corn,  oats,  etc.,  and  attempts  to  separate  the  mass  into 
its  constituents  by  picking  out  the  different  grains,  much 
difficulty  is  experienced  ;  but  if  the  handful  of  grain  be 
thrown  upon  a  large  flat  surface,  as  upon  a  table,  the 
grains  become  more  widely  separated  and  the  task  is 
considerably  simplified ;  or,  if  sown  upon  proper  soil 
the  various  grains  will  develop  into  growths  of  entirely 
different  external  appearance  by  which  they  can  readily 
be  recognized  as  unlike  in  nature.  Similarly,  if  a  test- 
tube  of  decomposed  bouillon  be  poured  out  upon  a  large 
flat  surface,  the  individual  bacteria  in  the  mass  are  very 
much  more  widely  separated  the  one  from  the  other 
than  they  were  when  the  bouillon  was  in  the  tube ;  but 
they  are  in  a  fluid  medium,  and  there  is  no  possibility 
of  their  either  remaining  separated  or  of  their  forming 
colonies  under  these  conditions,  so  that  it  is  impossible 
by  this  means  to  pick  out  the  individuals  from  the 
mixture. 


METHODS  OF  ISOLATION.  73 

If,  however,  it  is  possible  to  discover  some  substance 
which  possesses  the  property  of  being  at  one  time  fluid 
and  at  another  time  solid,  which  can  be  added  to  this 
bouillon  without  in  any  way  interfering  with  the  life- 
functions  of  the  bacteria,  then,  as  solidification  sets  in, 
the  organisms  will  be  fixed  in  their  positions  and  the 
conditions  will  be  analogous  to  that  seen  on  the  bit  of 
potato. 

Gelatin  possesses  this  property.  At  a  temperature 
which  does  not  interfere  with  the  life  of  the  organisms 
it  is  quite  fluid,  whereas  when  subjected  to  a  lower  tem- 
perature it  solidifies.  When  once  solid  it  may  be  kept 
at  a  temperature  favorable  to  the  growth  of  the  bacteria 
and  will  retain  its  solid  condition. 

Gelatin  was  added  to  the  fluids  containing  mixtures 
of  bacteria,  and  the  whole  was  then  poured  upon  a  large 
flat  surface,  allowed  to  solidify,  and  the  results  noted. 
It  was  found  that  the  conditions  seen  on  the  slice  of 
potato  could  be  reproduced,  that  the  individuals  in  the 
mixture  of  bacteria  grew  well  in  the  gelatin,  and,  as  on 
the  potato,  grew  in  colonies  of  typical  macroscopic  struc- 
ture, so  that  they  could  easily  be  distinguished  the  one 
from  the  other  by  their  naked-eye  appearances.  It  was 
necessary,  however,  to  use  a  more  dilute  mixture  of  bac- 
teria than  that  seen  in  the  original  decomposed  bouillon. 
The  number  of  individuals  in  the  tube  was  so  enormous 
that  on  the  gelatin  plate  they  were  so  closely  packed 
together  that  it  was  not  only  impossible  to  pick  them 
out  because  of  their  proximity  the  one  to  the  other,  but 
also  because  this  packing  together  materially  interfered 
with  the  production  of  those  characters  by  means  of 
which  differences  can  be  seen  with  the  naked  eye.  The 
numbers  of  organisms  were  then  diminished  by  a  process 


74  BACTERIOLOGY. 

of  dilution,  consisting  of  transferring  a  small  portion  of 
the  original  mixture  into  a  second  tube  of  sterilized 
bouillon  to  which  gelatin  had  been  added  and  liquefied ; 
from  this  a  similar  portion  was  added  to  a  third  gelatin- 
bouillon  tube,  and  so  on.  These  were  then  poured  upon 
large  surfaces  and  allowed  to  solidify.  The  results  were 
entirely  satisfactory.  On  the  gelatin  plates  from  the 
original  tube,  as  was  expected,  the  colonies  were  too 
numerous  to  be  of  any  use ;  on  the  plates  made  from 
the  first  dilution  they  were  much  fewer  in  number,  but 
still  they  were  usually  too  numerous  and  too  closely 
packed  to  permit  of  characteristic  growth ;  but  on  the 
second  dilution  they  were,  as  a  rule,  fewer  in  number 
and  widely  separated,  so  that  the  individuals  of  each 
species  were  in  no  way  prevented  by  the  proximity  of 
their  neighbors  from  growing  each  in  its  own  typical  way. 
There  was  then  no  difficulty  in  picking  out  the  colonies 
resulting  from  the  growth  of  the  different  individual 
bacteria. 

This,  then,  is  the  principle  underlying  Koch's  method 
for  the  isolation  of  bacteria  by  means  of  solid  media, 

The  fundamental  part  of  the  media  employed  is  the 
bouillon,  which  contains  all  the  elements  necessary  for 
the  nutrition  of  most  bacteria,  the  gelatin  being  em- 
ployed simply  for  the  purpose  of  rendering  the  bouillon 
solid.  The  medium  on  which  the  organisms  are 
growing  is,  therefore,  simply  solidified  bouillon,  or  beef 
tea. 

In  practice,  two  forms  of  gelatin  are  employed — the 
one  an  animal  or  bone  gelatin,  the  ordinary  table  gelatin 
of  good  quality ;  and  the  other  a  vegetable  gelatin, 
known  as  agar-agar,  or  Japanese  gelatin,  which  is  ob- 
tained from  a  group  of  algae  growing  in  the  sea  along 


METHODS  OF  ISOLATION.  75 

the  coast  of  Japan,  where  it  is  employed  as  an  article  of 
diet  by  the  natives. 

Aside  from  these  differences  in  origin  of  the  two  forms 
of  gelatin  employed,  their  behavior  toward  heat  and 
toward  bacteria  renders  them  of  different  application 
in  bacteriological  work.  The  animal  gelatin  liquefies 
at  a  much  lower  temperature,  and  also  requires  a  lower 
temperature  for  its  solidification,  than  does  the  agar- 
agar.  Ordinary  gelatin,  in  the  proportion  commonly 
used  in  this  work,  liquefies  at  about  24°-26°  C.,  and 
becomes  solid  at  from  8°-10°  C.  It  may  be  employed 
for  those  organisms  which  do  not  require  a  higher  tem- 
perature for  their  development  than  22°-24°  C.  Agar- 
agar,  on  the  other  hand,  does  not  liquefy  until  the  tem- 
perature has  reached  about  98°-99°  C.  It  remains  fluid 
ordinarily  until  the  temperature  has  fallen  to  38°-39° 
C.,  when  it  rapidly  solidifies.  For  our  purposes,  only 
that  form  of  agar-agar  can  be  used  which  remains  fluid 
at  from  38°-40°  C.  Agar-agar  which  remains  fluid 
only  at  a  temperature  above  this  point  would  be  too 
hot,  when  in  a  fluid  state,  for  use ;  many  of  the  organ- 
isms which  would  be  introduced  into  it  would  either  be 
destroyed  or  checked  in  their  development  by  so  high  a 
temperature.  Agar-agar  is  for  use  in  those  cases  in 
which  the  cultivation  must  be  conducted  at  a  tempera- 
ture above  the  melting  point  of  gelatin. 

In  addition  to  the  differences  toward  temperature, 
the  relations  of  these  two  gelatins  to  bacteria  are  differ- 
ent. Many  bacteria  bring  about  alterations  in  gelatin 
which  cause  it  to  become  liquid  (a  process  of  peptoniza- 
tion),  in  which  state  it  remains.  There  are  no  known 
organisms  that  bring  about  such  a  change  in  agar-agar. 

As  a  rule,  the  colony-formations  seen  upon   gelatin 


76  BACTERIOLOGY. 

are  much  more  characteristic  than  those  which  develop 
on  agar-agar,  and  for  this  reason  gelatin  is  to  be  pre- 
ferred when  circumstances  will  permit.  Both  gelatin 
and  agar-agar  may  be  used  in  the  preparation  of  plates 
and  Esmarch  tubes,  subsequently  to  be  described. 


CHAPTER   V. 

Preparation  of  media— Bouillon,  gelatin,  agar-agar,  potato,  blood-serum,  etc. 

As  has  been  stated,  the  fundamental  part  of  our  cul- 
ture media  is  beef  tea,  or  bouillon. 

BOUILLON. — The  directions  of  Koch  for  the  prepara- 
tion of  this  medium  have  undergone  many  modifications 
to  meet  special  cases,  but  for  general  use  his  original 
formula  is  still  retained.  It  is  as  follows  :  Five  hundred 
grammes  of  finely-chopped  lean  beef,  free  from  fat  and 
tendons,  is  to  be  soaked  in  one  litre  of  water  for  twenty- 
four  hours.  During  this  time  it  is  to  remain  in  the 
ice-chest  or  to  be  otherwise  kept  at  a  low  temperature. 
It  is  then  to  be  strained  through  a  coarse  towel  and 
pressed  until  a  litre  of  fluid  is  obtained.  To  this  is  to  be 
added  ten  grammes  (1  0  per  cent.)  of  dried  peptone  and 
five  grammes  (0.5  per  cent.)  of  common  salt  (ISaCl).  It 
is  then  to  be  rendered  exactly  neutral  or  very  slightly 
alkaline,  with  a  few  drops  of  saturated  sodium  carbon- 
ate solution.  The  flask  containing  the  mixture  is  then 
to  be  placed  either  in  the  steam  sterilizer  or  in  a  water- 
bath,  or  over  a  free  flame,  and  kept  at  a  boiling-point 
until  all  the  albumin  is  coagulated,  and  the  fluid  portion 
is  of  a  clear,  pale,  straw-color.  It  is  then  filtered 
through  a  folded  paper  filter,  and  sterilized  in  the  steam 
sterilizer  by  the  fractional  method.  Certain  of  the 
modifications  of  this  method  are  of  sufficient  value 
to  justify  mention.  Most  important  is  the  neutraliza- 


78  BACTERIOLOGY. 

tion.  Ordinarily,  this  is  accomplished  with  the  saturated 
sodium  carbonate  solution,  and  the  reaction  is  deter- 
mined with  the  ordinary  red  and  blue  litmus  papers. 

The  sodium  carbonate  solution  is  not  so  good  as  a 
strong  solution  of  caustic  soda  or  potash,  because  the 
carbonic  acid  liberated  from  the  sodium  carbonate  is 
frequently  seen  to  give  rise  to  confusing,  temporary  acid 
reaction  which  disappears  on  heating.  To  obviate  this, 
Schultz  (Centralbl  f.  Bald.  u.  Parasitenkunde,  1891, 
Bd.  x.,  Nos.  2  and  3)  recommends  exact  titration  with  a 
solution  of  caustic  soda.  For  this  purpose  a  4  per  cent, 
solution  of  caustic  soda  is  prepared.  From  this  a  0.4 
per  cent,  solution  is  made,  and  with  it  the  titration  is 
practised.  After  the  bouillon  has  been  deprived  of  all 
coagulable  albumin  and  blood-coloring  matter  by  boil- 
ing and  filtration,  and  has  cooled  down  to  the  temper- 
ature of  the  air,  its  whole  volume  is  exactly  measured. 

From  it  a  sample  of  exactly  5  or  10  c.c.  is  then  taken, 
and  to  this  a  few  drops  of  one  of  the  indicators  commonly 
employed  in  analytical  work  is  added.  Schultz  recom- 
mends 1  drop  of  phenolphthalein  solution  (1  gramme 
phenolphthalein  in  300  c.c.  of  alcohol)  to  1  c.c.  of  bouil- 
lon. The  beaker  containing  the  sample  is  placed  upon 
white  paper,  and  the  dilute  caustic  soda  solution  is  then 
allowed  to  drop  into  it,  very  slowly,  from  a  burette, 
until  there  appears  a  very  delicate  rose  color,  which 
indicates  the  beginning  of  alkaline  reaction.  A  second 
sample  of  the  bouillon  is  treated  in  the  same  way.  If 
the  amounts  of  caustic  soda  solution  required  for  each 
sample  deviate  but  very  slightly  or  not  at  all  the  one 
from  the  other,  the  mean  of  these  amounts  is  taken  as 
the  amount  of  alkali  necessary  to  neutralize  the  quantity 
of  bouillon  employed.  If  10  c.c.  of  bouillon  were  em- 


BOUILLON.  79 

ployed,  then,  for  the  whole  amount  of  1  litre,  just  100 
times  as  much,  minus  that  for  the  two  samples  used  in 
titration,  will  be  needed.  For  example  :  To  neutralize 
10  c.c.  of  bouillon,  2  c.c.  of  the  diluted  (0.4  per  cent.) 
caustic  soda  solution  were  employed.  For  the  remaining 
980  c.c.  of  the  litre  of  bouillon,  then,  196  c.c.  (200  c.c.— 
4  c.c.,  the  amount  employed  for  the  two  samples  of  10 
c.c.  each  of  bouillon)  is  needed  of  the  0.4  per  cent,  solution, 
or  one-tenth  of  this  amount  of  the  4  per  cent,  caustic  soda 
solution. 

For  the  neutralization  of  the  whole  bulk  of  the 
bouillon  it  is  better  to  employ  the  strong  alkaline 
solution,  as  by  its  use  the  volume  is  not  increased  to 
so  great  an  extent  as  when  the  dilute  solution  is  used. 

It  is  evident  that  this  method  is  much  more  exact 
than  that  ordinarily  employed,  but  at  the  same  time  it 
must  be  remembered  that  for  its  success  it  requires  ex- 
actness in  the  measurement  of  the  volumes,  and  the  prep- 
aration of  the  dilutions.  To  obviate  error,  it  is  better 
to  employ  this  method  when  the  solutions  are  all  cool 
and  of  nearly  the  same  temperature,  so  that  rapid  fluc- 
tuations in  temperature,  and  consequent  alterations  in 
volume,  will  not  materially  interfere  with  the  accuracy 
of  the  results. 

This  method  of  neutralization,  which  is  employed  by 
Schultz,  is  to  be  recommended  for  those  experiments  in 
which  slight  inaccuracies  in  the  reaction  of  the  media 
play  an  important  part. 

For  the  ordinary  purposes  of  the  beginner,  however, 
results  quite  satisfactory  in  their  nature  may  be  obtained 
by  the  employment  of  the  saturated  sodium  carbonate 
solution  for  neutralization  and  litmus  paper  as  the  indi- 
cator. For  some  time,  however,  it  has  been  our  practice 


80  BACTERIOLOGY. 

to  employ  the  yellow  curcuma  paper  for  the  detection  of 
alkalinity  rather  than  the  red  litmus  paper. 

Not  infrequently,  the  filtered  bouillon,  neutralized  and 
sterilized,  will  be  seen  to  contain  a  fine,  flocculent  pre- 
cipitate. This  may  be  due  either  to  excess  of  alkalinity 
or  to  incomplete  precipitation  of  the  albumin.  The 
former  may  be  corrected  with  dilute  acetic  or  hydro- 
chloric acid,  and  the  bouillon  again  boiled,  filtered,  and 
sterilized  ;  or,  if  due  to  the  latter  cause,  subsequent  boil- 
ing and  filtration  usually  results  in  ridding  the  bouillon 
of  the  precipitate. 

Another  modification  now  generally  employed  is  the 
use  of  meat  extracts  instead  of  the  infusion  of  meat. 
Almost  any  of  the  meat  extracts  of  commerce  answer 
the  purpose,  though  we  usually  employ  Liebig's.  It  is 
used  in  the  strength  of  from  two  to  four  grammes  to  the 
litre  of  water.  Peptone  and  sodium  chloride  are  added 
as  in  the  bouillon  made  from  the  meat  infusion.  The 
advantages  of  meat  extract  are :  It  takes  less  time ; 
aifords  a  solution  of  more  uniform  composition  if  used 
in  fixed  proportions,  and  in  general  use  gives  results 
that  are  equally  as  satisfactory  as  those  obtained  from 
the  employment  of  infusion  of  meat. 

NUTRIENT  GELATIN. — For  the  preparation  of  gela- 
tin the  bouillon  is  first  prepared  in  exactly  the  same 
way  as  has  just  been  described,  except  that  the  neutral- 
ization takes  place  after  the  gelatin  has  been  completely 
dissolved,  which  occurs  very  rapidly  in  hot  bouillon. 
The  reaction  of  the  gelatin  as  it  comes  from  the  manu- 
factories is  frequently  quite  acid,  so  that  a  much  larger 
amount  of  alkali  is  needed  for  its  neutralization  than  for 
other  media.  It  is  possible,  however,  to  obtain  from 
the  manufactories  an  excellent  grade  of  gelatin  from 


NUTRIENT  GELATIN. 


81 


which  all  acid  has  been  carefully  washed.1  The  gelatin 
is  added  in  the  proportion  of  10  to  12  per  cent.  Its 
complete  solution  may  be  accomplished  either  over  the 
water- bath,  in  the  steam  sterilizer,  or  over  a  free  flame. 
If  the  latter  method  be  practised,  care  must  be  taken 
that  the  mixture  is  constantly  stirred  to  prevent  burn- 
ing at  the  bottom  and  consequent  breaking  of  the  flask, 
if  a  flask  is  employed. 

For  some  time  it  has  been  our  practice  to  use,  for  the 
purpose  of  making  both  gelatin  and  agar-agar,  enamelled 
iron  saucepans  instead  of  glass  flasks ;  by  this  means  the 
free  flame  may  be  employed  without  danger  of  breaking 
the  vessel,  and,  with  a  little  care,  without  fear  of  burn- 
ing the  media.  Under  any  conditions  it  is  better  to 
protect  the  bottom  of  the  vessel  from  the  direct  action  of 
the  flame  by  the  interposition  of  several  layers  of  wire 
gauze,  a  thin  sheet  of  asbestos-board,  or  an  ordinary 
cast-iron  stove-plate. 

FIG.  13. 


When  the  gelatin  is  completely  melted,  it  may  be  fil- 
tered through  a  folded  paper  filter  on  an  ordinary  fun- 
nel ;  if  the  solution  is  perfect,  this  should  be  very  quickly 
accomplished. 

1  Hesteberg's  acid  free,  gold  label  gelatin  has  given  us  entire  satisfaction 
in  this  respect. 


82  BACTERIOLOGY. 

For  the  filtration  of  such  substances  as  gelatin  and 
agar-agar  it  is  of  much  importance  to  have  a  properly 
folded  filter.  To  fold  a  filter  correctly,  proceed  as  fol- 
lows :  A  circular  piece  of  filter  paper  is  folded  exactly 
through  its  centre,  forming  the  fold  1,1'  (Fig.  18)  ;  the 
end  1  is  then  folded  over  to  1',  forming  the  fold  5 ;  1 
and  V  are  each  then  brought  to  5,  thus  forming  the 
folds  3  and  7 ;  1  is  then  carried  to  the  point  7,  and  the 
fold  4  is  formed,  and  by  carrying  V  to  3  the  fold  6  is 
produced ;  and  by  bringing  1  to  3  and  1'  to  7  the  folds 
2  and  8  result. 

Thus  far  the  ridges  of  all  folds  are  on  the  side  of  the 
paper  next  to  the  table  on  which  we  are  folding.  The 
paper  is  now  taken  up,  and  each  space  between  the 
seams  just  produced  is  to  be  subdivided  by  a  seam  or 
fold  through  its  centre,  as  indicated  by  the  dotted  lines 
in  Fig.  13,  but  with  the  creases  on  the  side  opposite  to 

FIG.  14. 


that  occupied,  by  the  creases  1,  2,  3,  4,  etc.,  first  made. 
As  each  of  these  folds  is  made  the  paper  is  gradually 
folded  into  a  wedge-shaped  bundle  (Fig.  14  a),  which 
when  opened,  assumes  the  form  of  a  properly  folded 
filter  seen  in  6,  Fig.  14.  Before  placing  it  upon  the 
funnel  it  is  well  to  go  over  each  crease  again  and  see 
that  it  is  as  tightly  folded  as  possible,  without  tearing 


NUTRIENT  GELATIN.  83 

it.  The  advantage  of  the  folded  filter  is  that  by  its  use 
a  much  greater  filtering  surface  is  obtained,  as  it  is  in 
contact  with  the  funnel  only  at  the  points  formed  by  the 
ridges,  leaving  the  majority  of  the  flat  surface  free  for 
filtration. 

The  employment  of  the  hot-water  funnel,  so  often 
recommended,  has  been  dispensed  with  in  this  work 
to  a  very  large  extent,  as  we  know  that,  if  the  solution 
of  the  gelatin  is  complete,  filtration  is  so  rapid  as  not 
to  necessitate  the  use  of  an  apparatus  for  maintaining 
the  high  temperature.  The  temperature  at  which  the 
hot-water  funnel  retains  the  gelatin  is  so  high  that 
evaporation  and  concentration  rapidly  occur,  and  in  con- 
sequence the  filtration  is,  as  a  rule,  retarded.  The  filtra- 
tion is  frequently  done  in  the  steam  sterilizer,  but  this 
too  is  unnecessary  if  the  gelatin  is  quite  dissolved.  At 
the  ordinary  temperature  of  the  room,  and  by  the  means 
commonly  employed  for  the  filtration  of  other  sub- 
stances, both  gelatin  and  agar-agar  may  be  rapidly 
filtered  if  they  are  completely  dissolved. 

It  not  infrequently  occurs  that,  even  under  the  most 
careful  treatment,  the  filtered  gelatin  is  not  perfectly 
transparent  (the  condition  in  which  it  must  exist,  other- 
wise it  is  useless),  and  clarification  becomes  necessary. 
For  this  purpose  the  mass  must  be  redissolved,  and 
when  at  a  temperature  between  60°  C.  and  70°  C.,  the 
whites  of  two  eggs,  which  have  been  beaten  up  with 
about  50  c.c.  of  water,  are  added.  The  whole  is  then 
thoroughly  mixed  together  and  again  brought  to  the 
boiling-point,  and  kept  at  this  point  until  coagulation 
of  the  albumin  occurs.  It  is  better  not  to  break  up  the 
large  masses  of  coagulated  albumin  if  it  can  be  avoided, 


84  BACTERIOLOGY. 

as  when  broken  up  into  fine  flakes  they  clog  the  filter, 
and  materially  retard  filtration. 

The  practice  sometimes  recommended  of  removing 
these  albuminous  masses  by  first  filtering  the  gelatin 
through  a  cloth,  and  then  finally  through  paper,  is  not 
only  superfluous,  but  in  most  instances  renders  the  pro- 
cess of  filtration  much  more  difficult,  because  of  the  dis- 
integration of  these  masses  into  the  finer  particles,  which 
have  the  effect  just  mentioned,  viz.,  of  clogging  the  filter. 

Under  no  circumstances  is  a  filter  to  be  used  without 
first  having  been  moistened  with  water.  If  this  is  not 
done  the  pores  of  the  paper,  which  are  relatively  large 
when  in  a  dry  state,  when  moistened  by  the  gelatin  not 
only  diminish  in  size,  but  in  contracting  are  often 
entirely  occluded  by  the  finer  albuminous  flakes  which 
become  fixed  within  them,  and  filtration  practically 
ceases.  The  preliminary  moistening  with  water  causes 
the  diminution  of  the  size  of  the  pores  to  such  an  extent 
that  the  finer  particles  of  the  precipitate  rest  on  the 
surface  of  the  paper,  instead  of  becoming  fixed  in  its 
meshes. 

During  boiling  it  is  well  to  filter,  from  time  to  time, 
a  few  cubic  centimetres  of  the  gelatin  into  a  test-tube 
and  boil  it  over  a  free  flame  for  a  minute  or  so ;  in  this 
way  one  can  detect  if  all  the  albumin  has  been  coagu- 
lated, and  when  the  solution  is  ready  for  filtration. 

Gelatin  should  not,  as  a  rule,  be  boiled  over  ten  or 
fifteen  minutes  at  one  time,  or  left  in  the  steam  sterilizer 
for  more  than  thirty  to  forty-five  minutes,  otherwise  its 
property  of  solidifying  may  be  diminished. 

As  soon  as  the  gelatin  is  complete,  whether  it  is  re- 
tained in  the  flask  into  which  it  has  been  filtered  or  de- 
canted off  into  sterilized  test-tubes,  it  should  be  sterilized 


NUTRIENT  AGAR-AGAR.  85 

in  the  steam  sterilizer  on  three  successive  days,  for 
fifteen  minutes  each  day — the  mouth  of  the  flask  or  the 
test-tubes  containing  it  having  been  previously  closed 
with  cotton  plugs. 

NUTRIENT  AGAR-AGAR. — The  preparation  of  nutri- 
ent agar-agar  by  the  beginner  is  far  too  frequently  a 
tedious  and  time-taking  experience.  This  is  due  mainly 
to  lack  of  patience  and  deviation  from  the  rules  laid 
down  for  the  preparation  of  this  medium.  If  the  direc- 
tions given  below  for  the  preparation  of  nutrient  agar- 
agar  be  strictly  observed,  no  difficulty  whatever  should 
be  encountered.  Many  methods  are  recommended  for 
its  preparation ;  almost  every  worker  has  some  slight 
modification  of  his  own. 

The  methods  that  have  given  us  the  best  results,  and 
from  which  we  have  no  good  grounds  for  deviating,  are 
as  follows  : 

Prepare  the  bouillon  in  the  usual  way.  Agar-agar 
reacts  neutral  or  very  slightly  alkaline,  so  that  the 
bouillon  may  be  neutralized  before  the  agar-agar  is  added. 
Then  add  finely-chopped  or  powdered  agar-agar  in  the 
proportion  of  1  to  1.5  per  cent.  Place  the  mixture  in  a 
porcelain-lined  iron  vessel  and  make  a  mark  on  the 
sides  of  the  vessel  at  which  the  level  of  the  fluid  stands, 
add  about  250  c.c.  to  300  c.c.  of  water  and  allow  the 
mass  to  boil  slowly,  occasionally  stirring,  over  a  free 
flame,  for  from  one  and  one-half  to  two  hours.  Care 
must  be  taken  that  it  does  not  boil  over  the  sides  of  the 
vessel.  From  time  to  time  observe  if  the  fluid  has 
fallen  below  the  mark  of  its  original  level ;  if  it  has, 
add  water  until  its  original  volume  is  restored.  At  the 
end  of  the  time  given  remove  the  flame  and  place  the 
vessel  containing  the  mixture  in  a  large  dish  of  cold 

5 


86  BACTERIOLOGY. 

water ;  stir  the  agar-agar  continuously  until  it  has  cooled 
down  to  about  68°-70°  C.,  and  then  add  the  white  of 
one  egg  which  has  been  beaten  up  in  about  50  c.c.  of 
water ;  or  the  ordinary  dried  albumin  of  commerce  may 
be  dissolved  in  cold  water  in  the  proportion  of  about 
10  per  cent.,  and  used;  the  results  are  equally  as  good 
as  when  eggs  are  employed.  Mix  this  carefully  through- 
out the  agar-agar,  and  allow  the  mass  to  boil  slowly 
for  about  another  half-hour,  observing  all  the  while  the 
level  of  the  fluid.  It  is  necessary  to  reduce  the  tempera- 
ture of  the  mass  to  the  point  given,  68°-70°  C.,  other- 
wise the  coagulation  of  the  albumin  will  occur  suddenly 
in  lumps  and  masses  as  soon  as  it  is  added,  and  its  clear- 
ing action  will  not  be  homogeneous.  The  process  is 
purely  mechanical — the  finer  particles,  which  would 
otherwise  pass  through  the  pores  of  the  filter,  being 
taken  up  by  the  albumin  as  it  coagulates  and  retained 
in  the  coagula. 

At  the  end  of  one-half  hour  the  boiling  mass  may  be 
easily  and  quickly  filtered  through  a  heavy,  folded 
paper  filter  at  the  room  temperature,  and,  as  a  rule,  the 
filtrate  is  as  clear  and  transparent  as  agar-agar  usually 
ally  appears. 

It  might  be  well  to  emphasize  the  fact  that  for  the 
filtration  of  agar-agar  a  hot  water  funnel,  or  any  other 
special  device  for  maintaining  the  temperature  of  the 
mass  is  totally  unnecessary.  Agar-agar  prepared  after 
the  methods  just  given  should  filter  through  a  properly 
folded  paper  filter  at  the  rate  of  a  litre  in  from  twelve 
to  fifteen  minutes. 

Another  plan  that  insures  complete  solution  of  the  agar- 
agar  without  causing  the  precipitates  that  are  commonly 
seen  when  all  the  ingredients  are  added  at  first  and 


NUTRIEN1  AQAE-AGAE.  87 

boiled  for  a  long  time,  is  to  weigh  out  the  necessary 
amount  of  agar-agar,  10  or  15  grammes,  and  place  this 
in  1300  or  1400  c.c.  of  water  and  boil  down  over  a  free 
flame  to  1000  c.c.  The  peptone,  salt,  and  beef-extract 
are  then  added  and  the  boiling  again  continued  until 
they  are  dissolved.  The  clarification  with  egg  albumin 
may  then  be  done,  and  usually  the  mass  filters  quite 
clear  and  does  not  show  the  presence  of  precipitates 
upon  cooling.  If  the  mixture  is  positively  alkaline,  it 
is  not  only  cloudy  but  it  filters  with  difficulty ;  if  it  is 
acid,  it  is  usually  quite  clear,  filters  more  quickly,  but, 
as  Schulze  has  pointed  out,  loses  at  the  same  time  some 
of  its  gelatinizing  properties.  The  bouillon  should 
always  be  neutralized  before  the  agar-agar  is  added  to 
it,  for  if  the  bouillon  be  acid,  from  the  acid  of  the  meat, 
it  robs  the  agar-agar,  under  the  influence  of  heat,  of 
some  of  its  gelatinizing  powers,  which  cannot  be  re- 
gained by  subsequent  neutralization. 

Another  method  by  which  the  agar-agar  can  easily 
and  quickly  be  melted,  is  by  steam  under  pressure.  If 
the  flask  containing  the  mixture  of  bouillon  and  agar- 
agar  be  kept  in  the  digester  or  autoclave,  with  the  steam 
under  a  pressure  of  about  one  atmosphere,  as  shown 
by  the  gauge,  for  ten  minutes,  the  agar-agar  will  be 
found  at  the  end  of  this  time  completely  melted,  and 
filtration  may  then  be  accomplished  with  but  little  diffi- 
culty. 

If  glycerin  is  to  be  added  to  the  agar-agar,  it  is  done 
after  filtration  and  before  sterilization.  The  nutritive 
properties  of  the  media  for  certain  organisms,  particu- 
larly the  tubercle  bacillus,  is  improved  by  the  addition 
of  glycerin  in  the  proportion  of  5  to  7  per  cent. 

If  after  filtration  a  fine  flocculent  precipitate  is  seen, 


88  BACTERIOLOGY. 

look  to  the  reaction  of  the  medium.  If  it  is  quite  alka- 
line, neutralize,  boil,  and  filter  again.  If  the  reaction 
is  neutral  or  only  very  slightly  acid,  dissolve  and  clarify 
again  with  egg  albumin  by  the  method  given. 

The  most  important  point  in  all  the  media,  aside 
from  the  correct  proportion  of  the  ingredients,  is  their 
reaction.  They  must  be  neutral  or  very  slightly  alka- 
line. Only  a  few  organisms  develop  well  on  media  of 
an  acid  reaction.  In  all  of  the  above  media  the  meat 
extracts  now  on  the  market  may  usually  be  substituted 
for  the  meat  itself  in  preparing  the  bouillon.  They  may 
be  employed  in  the  proportion  of  from  two  to  four 
grammes  to  the  litre  of  water. 

PREPARATION  OF  POTATOES. — Potatoes  are  prepared 
for  use  in  two  ways  : 

1.  They  are  taken  as  they  come  to  the  market — old 
potatoes  being  usually  recommended,  and  carefully 
scrubbed  under  the  water-tap  with  a  stiff  brush  until 
all  adherent  dirt  has  been  removed  ;  "  the  eyes  "  and  all 
discolored  or  decayed  parts  are  carefully  removed  with 
a  pointed  knife.  They  are  then  to  be  placed  in  a  solu- 
tion of  corrosive  sublimate  of  the  strength  of  1  : 1000 
and  allowed  to  remain  there  for  twenty  minutes ;  at  the 
eud  of  this  time,  without  rinsing  off  the  sublimate,  they 
are  placed  in  a  covered  tin  bucket  with  a  perforated 
bottom  and  sterilized  in  the  steam  sterilizer  for  forty- 
five  minutes.  On  the  second  and  third  days  the  sterili- 
zation is  repeated  for  fifteen  to  twenty  minutes  each 
day.  They  must  not  be  removed  from  the  sterilizing 
bucket  until  sterilization  is  complete.  At  the  end  of 
this  time  they  are  ready  for  use.  When  prepared  in 
this  way,  they  are  usually  intended  to  be  cut  in  half, 


PREPARATION  OF  POTATOES.  89 

and  the  cultivation  of  the  organisms  is  to  be  con- 
ducted upon  the  flat  surfaces  of  the  sections. 

This  method  requires  some  care  to  prevent  contam- 
ination during  manipulation.  The  hand  which  is  to 
take  up  the  potato  from  the  bucket,  which  until  now  has 
remained  covered,  is  first  disinfected  in  the  sublimate 
solution  for  ten  minutes,  the  potato  is  then  taken  up 
between  the  thumb  and  index  finger,  and  severed  into 
two  by  a  knife  which  has  just  been  sterilized  in  the  free 
flame  until  it  is  quite  hot.  The  blade  of  the  knife  is 
passed  not  quite  through  the  potato,  but  nearly  so.  A 
large  glass  culture-dish  for  the  reception  of  the  two 
halves  of  the  potato  having  been  disinfected  for  twenty 
minutes  with  1:1000  sublimate  solution  and  then 
drained  of  all  the  adherent  solution,  is  at  hand  ready 
for  the  bits  of  potato ;  the  cover  is  removed,  and  by 
twisting  the  knife  gently  the  two  halves  of  the  potato 
may  be  caused  to  fall  apart  in  the  dish  and  usually  to 
fall  upon  their  convex  surfaces,  leaving  the  flat  sections 
uppermost.  The  cover  is  placed  upon  the  dish  and  the 
potatoes  are  ready  for  inoculation. 

2.  Preparation  of  potatoes  for  test-tube  cultures.  Method 
of  Bolton1.  If  the  potatoes  are  to  be  employed  for  test- 
tube  cultures,  one  simply  scrubs  off  the  coarser  particles 
of  dirt  with  water  and  a  brush,  and  with  a  cork-borer 
punches  out  cylindrical  bits  of  potato  which  will  fit 
loosely  into  the  test-tubes  to  be  used.  On  each  bit  of 
potato  is  then  to  be  cut  a  slanting  surface  running  from 
about  the  junction  of  the  first  and  second  third  of  the 
cylinder  to  the  diagonally  opposite  end.  These  cylin- 
ders of  potato  are  now  to  be  left  in  running  water  over 
night,  otherwise  they  are  very  much  discolored  by  the 

1  Medical  News,  1887,  vol.  i.  p.  138. 


90 


BACTERIOLOGY. 


FIG.  15. 


sterilization  to  which  they  are  to  be  subjected.  At  the 
end  of  this  time  they  are  placed  in  previously  prepared 
test-tubes,  one  piece  in  each  tube,  with 
the  slanting  surface  up,  the  cotton  plugs 
of  the  tubes  replaced,  and  they  are  then 
to  be  sterilized  iu  the  steam  for  forty- five 
minutes.  On  the  second  and  third  days 
they  are  to  be  sterilized  for  fifteen  to 
twenty  minutes  each  day. 

Or  the  entire  sterilization  may  be  ac- 
complished in  the  autoclave  with  the 
steam  under  a  pressure  of  one  atmosphere, 
by  a  single  exposure  of  twenty  to  twenty- 
five  minutes.  When  finished  they  have 
the  appearance  seen  in  Fig.  15,  except 
that  there  is  no  growth  upon  the  surface 
as  is  shown  in  the  cut. 

For  some  purposes  potatoes  may  be  ad- 
vantageously peeled,  sliced  into  discs  of 

potato  iT^st-tube.  about  l  c-m-  in  thickness,  and  placed  in 
small  glass  dishes  provided  with  covers, 
similar  to  the  ordinary  Petri  dishes.  The  dish  and  its 
contents  are  then  sterilized  by  steam  in  the  usual  way 
(method  suggested  by  von  Esmarch).  By  this  plan  a 
relatively  large  area  for  cultivation  is  obtained. 

Potatoes  may  also  be  boiled,  or  steamed,  and  mashed, 
and  the  mass  placed  in  covered  dishes,  test-tubes,  or 
flasks,  and  sterilized.  By  this  method  one  obtains  in 
the  mass  a  mean  of  the  composition  of  the  several  pota- 
toes, or  bits  of  potatoes,  used  in  making  it,  an  advantage 
where  uniformity  is  desired. 

Care  must  be  given  to  the  sterilization  of  potatoes, 
because  they  always  have  adhering  to  them  the  organ- 


BLOOD-SERUM.  91 

isms  commonly  found  in  the  ground,  the  spores  of  which 
are  among  the  most  resistant  known.  The  so-called 
"  potato  bacillus  "  is  one  of  this  group ;  it  is  an  organ- 
ism which  is  not  infrequently  more  or  less  of  an  obstacle 
to  the  work  of  the  beginner. 

BLOOD-SERUM.  —  Originally  blood-serum  required 
special  care  in  its  preparation  ;  it  was  always  necessary 
to  reduce  the  unavoidable  contamination,  which  to  a 
certain  extent  occurs  when  the  blood  is  obtained,  to  its 
minimum  degree. 

It  is  possible  to  collect  serum  from  small  animals  and 
in  small  quantities  under  such  precautions  that  it  is 
perhaps  not  contaminated,  but,  ordinarily,  for  laboratory 
purposes  a  larger  quantity  is  needed,  so  that  the 
slaughter-houses  form  the  sources  from  which  it  is 
usually  obtained,  and  here  a  certain  amount  of  contam- 
ination is  unavoidable,  though  its  degree  may  be  limited 
by  proper  precaution. 

The  steps  that  were  formerly  thought  to  be  essential 
to  the  successful  collection  of  blood  and  the  preparation 
of  serum  for  culture  purposes  were  about  as  follows : 

The  animal  from  which  the  blood  is  to  be  collected 
should  be  drawn  up  to  the  ceiling  by  the  hind  legs,  the 
head  should  be  held  well  back,  and  with  one  pass  of  a  very 
sharp  knife  the  throat  should  be  completely  cut  through. 
The  blood  which  spurts  from  the  severed  vessels  should 
be  collected  in  large  glass  jars  which  have  been  previously 
cleaned,  disinfected,  and  all  traces  of  the  disinfectant  re- 
moved with  alcohol  and,  finally,  ether.  The  latter  evap- 
orates very  quickly  and  leaves  the  jar  quite  dry.  The 
jars  should  be  provided  with  covers  which  close  her- 
metically— these,  too,  should  be  carefully  disinfected. 
The  best  form  of  glass  vessels  for  the  purpose  is  the 


92  BACTERIOLOGY. 

large  glass  museum  jar  of  about  one  gallon  capacity, 
which  closes  by  a  cover  that  can  be  tightly  screwed 
down  upon  a  rubber  joint.  From  two  such  jarfuls  of 
blood  one  can  recover  quite  a  large  quantity  of  clear 
serum,  ordinarily  from  500-700  c.c.  The  jars  having 
been  filled  with  blood,  their  covers  are  placed  loosely 
upon  them  and  they  are  allowed  to  stand  for  about 
fifteen  minutes  until  clotting  has  begun.  At  the  end 
of  this  time  a  clean  glass  rod  is  passed  around  the  edges 
of  the  surface  of  the  clot  to  break  up  any  adhesions  to 
the  wall  of  the  jar  that  might  have  formed,  and  which 
would  prevent  the  sinking  of  the  clot  to  the  bottom. 
The  covers  are  then  tightly  replaced,  and  with  as  little 
agitation  as  possible  the  jars  are  placed  in  an  ice-chest, 
where  they  remain  for  twenty-four  to  forty-eight  hours. 
The  temperature  should,  however,  not  be  low  enough 
to  prevent  coagulation,  but  should  be  sufficiently  low  to 
interfere  with  the  development  of  any  living  organisms 
that  may  be  present.  The  temperature  of  the  ordinary 
domestic  refrigerator  is  sufficient  for  the  purpose.  After 
twenty-four  to  forty-eight  hours  the  clot  will  have  be- 
come firm,  and  will  be  seen  at  the  bottom  of  the  jar. 
Above  it  is  a  quantity  of  dark  straw-colored  serum. 
The  serum  may  then  be  drawn  off  with  a  sterilized 
pipette  and  placed  in  tall  cylinders  that  have  previ- 
ously been  plugged  with  cotton  wadding  and  sterilized. 
After  treating  all  the  serum  in  this  way,  care  having 
been  taken  to  get  as  little  of  the  coloring  matter  of  the 
blood  as  possible,  it  may  be  placed  again  in  the  ice- 
chest  for  twenty-four  hours,  during  which  time  the  cor- 
puscular elements  will  sink  to  the  bottom,  leaving  the 
supernatant  fluid  quite  clear.  This  may  then  be  pipetted 
off,  either  into  sterilized  test-tubes,  about  8  c.c.  to  each 


BLOOD-SERUM. 


93 


tube,  or  into  small  sterilized  flasks  of  about  100  c.c. 
capacity.  It  is  then  to  be  sterilized  by  the  intermittent 
method  at  low  temperatures,  viz.,  for  one  hour  on  each 
of  five  consecutive  days  at  a  temperature  of  68°-70°  C. 
During  the  intervening  days  it  is  to  be  kept  at  the  room 
temperature  to  permit  of  the  development  of  any  spores 
that  maybe  present  into  their  vegetative  forms,  in  which 
condition  they  are  killed  by  an  hour's  exposure  to  the 
temperature  of  70°  C. 

At  the  end  of  this  time  the  serum  in  the  tubes  may 

FIG.  16. 


Chamber  for  sterilizing  and  solidifying  blood-serum.    (Kocn.) 

either  be  retained  as  fluid  serum  or  solidified  at  between 
76°-80°  C.  In  solidifying  the  serum  the  tubes  should 
be  placed  in  an  inclined  position  so  that  as  great  a 
surface  as  possible  may  be  given  to  the  serum.  The 
process  of  solidification  requires  constant  attention  if 
good  results  are  to  be  obtained,  i.  e.,  if  a  translucent, 
solid  medium  is  to  result.  If  the  old,  small  form  of 
apparatus  be  employed  (Fig.  16),  then  the  solidification 


OF  THE 
UNIVERSITY 


94  BACTERIOLOGY. 

can  be  accomplished  in  a  shorter  time  than  if  the  larger 
forms,  which  are  now  frequently  employed,  are  used. 
No  definite  rule  for  the  time  that  will  be  required  can 
be  laid  down,  for  this  is  not  constant.  If  the  small 
solidifying  apparatus  be  used,  very  good  results  may  be 
obtained  in  about  two  hours  at  78°  C.  It  frequently 
requires  a  longer  time  at  a  higher  temperature  than 
has  been  mentioned.  This  is  especially  the  case  with 
Loeffler's  serum  mixture. 

The  best  results  are  obtained  when  a  low  temperature 
is  employed  for  a  long  time.  Under  any  circumstances 
the  tubes  must  be  observed  from  time  to  time  through 
the  glass  door  or  cover  with  which  the  solidifying  oven 
is  provided,  and  each  time  the  oven  should  be  slightly 
jarred  with  the  hand  to  see  if  solidification,  as  indicated 
by  the  disappearance  of  tremors  from  the  serum,  is  be- 
ginning. If  the  temperature  gets  too  high,  or  the  ex- 
posure is  too  long,  an  opaque  medium  results.  The 
temperature  to  be  observed  is  that  of  the  air  inside  the 
chamber,  and  also  that  of  the  water  surrounding  it. 
The  latter  is  usually  a  degree  or  two  higher  than  the 
former.  The  tubes  should  not  rest  directly  upon  the 
heated  bottom  or  against  the  heated  sides  of  the  chamber, 
but  should  lie  upon  racks  of  wood  or  wire,  and  be  pro- 
tected from  the  sides  by  a  wire  screen  of  gauze  ;  in  this 
way  the  tubes  are  all  exposed  to  about  the  same  tem- 
perature. The  thermometer  which  indicates  the  tem- 
perature inside  the  chamber  should  not  touch  the  sur- 
faces, but  should  either  be  suspended  free  from  above 
through  a  cork  in  the  top  of  the  apparatus,  if  the  large 
form  of  apparatus  be  used,  or  should  lie  upon  a  rack  of 
cork  or  wood,  its  bulb  being  free  and  a  little  lower  than 
the  other  extremity,  if  the  small,  old-fashioned  appa- 


BLOOD-SERUM.  95 

ratus  of  Koch  be  employed.     The  latter  form  is  prefer- 
able, as  it  is  more  easily  managed. 

When  solidification  is  complete,  the  tubes  are  to  be 
retained  in  the  erect  position,  and,  unless  they  are  in- 
tended for  immediate  use,  must  be  prevented  from  dry- 
ing. The  superfluous  ends  of  the  cotton  plugs  should 
be  burned  off,  and  the  mouths  of  the  tubes  may  then  be 
covered  by  sterilized  rubber  caps,  or,  as  Ghriskey  sug- 
gests, they  may  be  closed  with  sterilized  corks  pushed 
in  on  top  of  the  cotton  plugs.  Even  with  the  greatest 
care  it  not  uncommonly  happens  that  one  or  two  of  the 
lot  of  tubes  thus  prepared  and  protected  will  become 
contaminated.  This  is  usually  due  to  spores  of  moulds 
that  have  fallen  into  the  rubber  caps  or  on  the  cotton 
plugs  during  manipulation,  and,  finding  no  means  of 
outward  growth,  have  thrown  their  hyphse  downward 
through  the  cotton  into  the  tube,  and  their  spores  have 
fallen  on  the  surface  of  the  serum  and  developed  there. 

The  foregoing  is,  in  the  main,  the  plan  originally 
recommended  by  Koch  for  the .  preparation  of  this 
medium.  In  recent  times,  however,  particularly  since 
the  study  of  diphtheria  by  the  method  of  Loeffler  has 
become  so  general,  and  large  quantities  of  serum  tubes 
were  found  to  be  necessary,  a  modification  has  been  sug- 
gested that  has,  in  this  country  at  least,  almost  entirely 
supplanted  the  method  by  Koch.  The  popularity  of 
the  Councilman-Mallory  method  is  due  to  the  following 
facts  :  By  it  the  serum  is  more  quickly  and  easily  pre- 
pared ;  rigid  precautions  against  contamination  during 
collection  of  the  serum  are  not  so  necessary,  and  the 
resulting  medium,  while  not  transparent  or  even  trans- 
lucent (points  aimed  at  in  the  original  method),  fully 
meets  all  the  requirements. 


96  BACTERIOLOGY. 

The  special  points  in  the  method  are  :  The  serum  is 
decanted  into  test  tubes  as  soon  as  obtained  ;  it  is  then 
firmly  coagulated  in  a  slanting  position  in  the  dry  air 
sterilizer  at  from  80°  to  ^0°  C. ;  it  is  then  sterilized  in 
the  steam  sterilizer  at  100°  C.  on  three  successive  days, 
as  in  the  case  of  other  culture  media.  It  may  then  be 
protected  against  evaporation  by  sterilized  rubber  caps 
or  sterilized  corks  in  the  way  already  described,  and  set 
aside  until  needed. 

Unless  the  coagulation  in  the  dry  sterilizer  be  com- 
plete, the  surface  of  the  serum  will  be  found  to  be  lacer- 
ated by  bubbles  and  cavities  after  it  has  been  subjected 
to  the  steam  sterilization.  A  similar  formation  of  cavi- 
ties over  the  surface  of  the  serum  will  occur  if  the  tem- 
perature of  the  hot  air  sterilizer,  in  which  it  is  solidified, 
is  allowed  to  get  above  90°  C.,  or  if  it  be  elevated  to 
this  point  too  quickly. 

It  is  of  no  special  advantage  to  have  the  serum  clear, 
as  the  admixture  of  blood-coloring  matter  does  not  aifect 
its  nutritive  properties. 

It  is  often  desirable  to  obtain  blood-serum  in  small 
quantities,  either  for  culture  purposes  or  for  the  study 
of  the  serum  of  different  animals  in  its  relation  to  bac- 
teria, and  for  this  purpose  Nuttall  (Centralbl.  fur  Bald,  u 
Parasitenkunde,  1892,  Bd.  xi.  p.  539)  suggests  a  very 
convenient  method.  By  the  use  of  a  sterilized  vessel, 
of  the  shape  given  in  Fig.  17,  from  ten  to  one  hundred 
cubic  centimetres  of  blood  can  be  collected,  and  if  proper 
precautions  are  observed  no  contamination  by  bacteria 
need  occur.  The  collecting  bulb  is  used  in  the  follow- 
ing way :  An  artery,  either  femoral  or  carotid,  is  ex- 
posed, and  around  it  two  ligatures  are  placed  ;  that 
distant  from  the  heart  is  tightened,  while  the  one  nearest 


BLOOD-SERUM.  97 

the  heart  is  left  loose ;  between  the  latter  and  the  heart 
the  artery  is  clamped.  A  small  slit  is  then  made  in  its 
wall,  into  which  the  point  a  of  the  bulb  is  introduced 
and  the  artery  bound  tightly  around  it  with  the  hitherto 
loose  ligature;  the  clamp  is  removed  and  the  bulb 

FIG.  17. 


a 
Nuttall's  bulb  for  collecting  blood-serum  under  antiseptic  precautions. 

quickly  fills  with  blood.  The  clamp  is  now  again  put 
in  position,  the  point  of  the  bulb  removed  and  sealed 
in  the  gas- flame,  the  loose  ligature  tightened,  the  wound 
closed,  and  the  vessel  containing  the  blood  is  set  aside 
in  a  cool  place  until  coagulation  has  occurred.  The 
serum  is  most  easily  withdrawn  from  the  bulb  by  means 
of  a  pipette,  closed  above  with  a  cotton  plug,  and  sup- 
plied with  a  bit  of  rubber  tubing,  about  one-half  metre 
long,  with  glass  mouth-piece.  By  holding  the  pipette  in 
the  hand  and  sucking  upon  the  rubber  tube  one  can 
more  easily  direct  the  point  of  the  pipette  than  if  it  is 
used  in  the  ordinary  way. 

The  bulbs  are  easily  blown,  and  after  having  been 


98  BACTERIOLOGY. 

sealed  at  the  point  and  plugged  with  cotton  can  be  kept 
on  hand  just  as  are  sterilized  test-tubes. 

It  is  sometimes  desirable  to  preserve  blood-serum  in  a 
fluid  state.  This  can  be  done  by  the  fractional  method 
of  sterilization  at  low  temperatures,  already  described, 
or  with  much  less  effort,  and  without  the  use  of  heat  by 
a  method  that  we  have  found  to  be  very  satisfactory. 
In  the  studies  of  Kirchner,  chloroform  was  shown  to 
possess  decided  disinfectant  properties ;  as  it  is  quite 
volatile  it  is  easily  removed  when  its  disinfectant  or  an- 
tiseptic functions  are  no  longer  required.  If,  therefore, 
the  serum  to  be  preserved  be  placed  in  a  closely- 
stoppered  flask  and  enough  chloroform  added  to  form  a 
thin  layer,  about  2  mm.  on  the  bottom,  the  serum  may 
be  kept  indefinitely  without  contamination,  so  long  as 
the  chloroform  is  not  permitted  to  evaporate.  When 
required  for  use  the  serum  is  decanted  into  test-tubes, 
which  are  then  placed  in  a  water-bath  at  about  50°  C. 
until  all  the  chloroform  has  been  driven  off;  this  can  be  ' 
determined  by  the  disappearance  of  its  characteristic 
odor.  The  serum  may  then  be  solidified,  sterilized  by 
heat,  and  employed  for  culture  purposes.  We  have 
found  serum  so  preserved  to  answer  all  requirements 
as  a  culture  medium. 

SPECIAL  MEDIA. — The  media  just  described — bou- 
illon, nutrient  gelatin,  nutrient  agar-agar,  potato,  and 
blood-serum — are  those  in  general  use  in  the  laboratory 
for  purposes  of  isolation  and  study  of  the  ordinary  forms 
of  bacteria.  For  the  finer  points  of  differentiation 
special  media  have  been  suggested  ;  a  few  of  them  will 
be  mentioned. 

Milk.  Fresh  milk  should  be  allowed  to  stand  over 
night  in  the  ice-chest,  the  cream  then  removed,  and  the 


SPECIAL  MEDIA.  99 

remainder  of  the  milk  pipetted  into  test-tubes,  about 
8  c.c.  to  each  tube,  and  sterilized  by  the  intermittent 
process,  at  the  temperature  of  steam,  for  three  successive 
days. 

The  separation  of  the  cream  may  be  accelerated  and 
rendered  more  complete  by  one  sterilization  of  the  milk 
in  the  cylinder  before  it  is  placed  in  the  ice  chest. 

The  cream  is  best  separated  from  the  milk  by  the  use 
of  a  cylindrical  vessel  with  stopcock  at  the  bottom, 
by  means  of  which  the  milk,  devoid  of  cream,  may  be 
drawn  off.  A  Chevalier  creamometer  with  stopcock  at 
the  bottom  serves  the  purpose  very  well.  It  should  be 
covered  while  standing. 

Milk  may  be  used  as  a  culture  medium  without  any 
addition  to  it,  or,  before  sterilizing,  a  few  drops  of 
litmus  tincture  may  be  added,  just  enough  to  give  it  a 
pale  blue  color.  By  this  means  it  may  be  seen  that  dif- 
ferent organisms  bring  about  different  reactions  in  the 
medium  ;  some  producing  alkalies  which  cause  the  blue 
color  to  be  intensified,  others  producing  acids  which 
change  it  to  red,  while  others  bring  about  neither  of 
these  changes.  Similarly  litmus  solution  is  often  added 
to  gelatin  and  agar-agar  for  the  same  purpose. 

Milk  may  also  be  employed  as  a  solid  culture  medium 
by  the  addition  to  it  of  gelatin  or  agar-agar  in  the  pro- 
portions given  for  the  preparation  of  the  ordinary  nutri- 
ent gelatin  or  agar-agar.  It  has,  however,  in  this  form 
the  disadvantage  of  not  being  transparent,  and  can  there- 
fore best  be  used  for  the  study  of  those  organisms  which 
grow  upon  the  surface  of  the  medium  without  causing 
liquefaction. 

Nutrient  gelatin  and  agar-agar  can  also  be  prepared 


100  BACTERIOLOGY. 

from  neutral  milk  whey,  obtained  from  milk  after  pre- 
cipitation of  the  casein. 

Dunham's  peptone  solution.  The  medium  usually 
known  as  Dunham's  solution  is  prepared  according  to 
the  following  formula  : 

Dried  peptone  .       .       .       .       ,       .       ...       1     part. 

Sodium  chloride 0.5    " 

Distilled  water . 100     parts. 

It  is  usually  of  a  neutral  or  slightly  alkaline  reaction, 
and  neutralization  is  not,  therefore,  necessary.  It  is 
filtered,  decanted  into  tubes  or  flasks,  and  sterilized  in 
the  steam  sterilizer  in  the  ordinary  way.  The  most 
common  use  to  which  this  solution  is  put  is  in  deter- 
mining if  the  organism  under  consideration  possesses 
the  property  of  producing  indol  as  one  of  its  products 
of  nutrition.  It  is  essential  for  accuracy  that  the  prep- 
aration of  dried  peptone  employed  should  be  of  as 
nearly  chemical  purity  as  is  possible,  and  indeed  the 
other  ingredients  should  be  correspondingly  free  from 
impurities,  Gorini  (Centralblatt  fur  Balderiologie  und 
Parasitenkunde,  1893,  Bd.  xiii.  p.  790)  calls  attention 
to  the  fact  that  impurities  in  the  peptone,  particularly 
the  presence  of  carbohydrates,  so  interfere  with  the 
production  of  indol  by  certain  bacteria  that  otherwise 
produce  it,  that  it  is  ofttimes  impossible,  when  such  prep- 
arations have  been  employed,  to  obtain  the  characteristic 
color  reaction  of  this  body,  and  where  it  is  obtained  it 
is  always  after  a  much  longer  time  than  is  the  case 
where  peptone  free  from  these  substances  has  been  used. 
He  suggests  the  advisability  of  testing  the  purity  of  all 
peptone  preparations  before  using  them,  by  means  of 
the  reaction  that  they  exhibit  when  acted  upon  by 
Fehling's  alkaline  copper  solution.  Under  the  influence 


SPECIAL  MEDIA.  101 

of  this  agent,  pure  peptone  in  solution  gives  a  violet 
color  (the  biuret  reaction),  which  remains  permanent 
even  after  boiling  for  five  minutes.  If  instead  of  a 
violet  color  there  appears  a  red  or  reddish-yellow  pre- 
cipitate the  peptone  should  be  discarded,  as  in  his  ex- 
perience no  indol  is  produced  from  peptone  giving  this 
reaction.  Both  the  peptone  solution  and  that  of  the 
copper  (particularly  the  latter)  should  be  relatively 
dilute  in  order  for  the  reaction  to  be  successful. 

Peptone  rosalic  acid  solution.  Peptone  solution  con- 
taining rosalic  acid  serves  well  for  the  detection  of  altera- 
tions in  reaction.  It  consists  of  the  peptone  solution 
of  Dunham,  to  each  100  c.c.  of  which  2  c.c.  of  the  fol- 
lowing solution  is  added  : 

Rosalie  acid  (coralline) 0.5  gramme. 

Alcohol  (80  per  cent.) 100     c.c. 

This  is  to  be  boiled,  filtered,  and  decanted  into  clean, 
sterilized  test-tubes,  about  8  to  10  c.c.  to  each  tube.  The 
tubes  are  then  to  be  sterilized  in  the  usual  way  by  steam. 
When  sterilization  is  completed  and  the  tubes  cooled,  the 
solution  will  be  of  a  very  pale  rose  color,  which  disap- 
pears entirely  under  the  action  of  acids,  and  becomes 
much  more  intense  when  alkalies  are  produced.  We 
have  used  this  solution  for  some  time  for  the  study  of 
the  reactions  produced  by  different  organisms,  and  find 
it  a  valuable  addition  to  our  means  of  differentiating 
bacteria. 

Rosalic  acid  cannot  be  used  with  safety  in  solutions 
containing  glucose,  as  the  reducing  action  of  the  latter 
deprives  it  of  its  color. 

Lactose-litmus-agar,  or  gelatin  of  Wurtz.  A  medium 
of  much  use  in  the  differentiation  of  bacteria  is  that 


102  BACTERIOLOGY. 

recommended  by  Wurtz,  consisting  of  ordinary  nutrient, 
slightly  alkaline  agar-agar,  to  which  from  2  to  3  per 
cent,  of  lactose  and  sufficient  litmus  tincture  to  give  it 
a  pale  blue  color  have  been  added.  Bacteria  capable  of 
causing  fermentation  of  lactose  when  grown  on  this 
medium  develop  into  colonies  of  a  pale  pink  color  and 
cause,  likewise,  a  reddening  of  the  surrounding  medium, 
owing  to  the  production  of  acid  as  a  result  of  their 
action  upon  the  lactose  ;  while  other  bacteria,  incapable 
of  such  fermentative  activities,  grow  as  pale  blue  colonies 
and  cause  no  reddening  of  the  surrounding  medium. 
It  is  especially  useful  for  the  differentiation  of  the 
bacillus  of  typhoid  fever,  which  does  not  possess  the 
property  of  bringing  about  fermentation  of  lactose,  from 
other  organisms  that  simulate  it  in  many  other  respects 
but  which  do  possess  this  property. 

Its  preparation  is  as  follows^:  To  nutrient  agar-agar 
or  gelatin,  the  alkalinity  of  which  is  such  that  one  cubic 
centimetre  will  require  0.1  c.c.  of  a  1  :  20  normal  sul- 
phuric acid  solution  to  neutralize  it,  lactose  is  added  in 
the  proportion  of  2  or  3  per  cent.  ;  it  is  then  decanted 
into  test-tubes  and  sterilized  in  the  usual  way.  When 
sterilization  is  complete  there  is  to  be  added  to  each 
tube  enough  sterilized  litmus  tincture  to  give  a  decided 
though  not  very  intense  blue  color.  This  must  be  done 
carefully,  to  avoid  contamination  of  the  tubes  during 
manipulation.  It  is  better  not  to  add  the  litmus  tinc- 
ture before  sterilizing  the  tubes,  as  its  color  character- 
istics are  in  some  way  altered  by  its  contact  with  organic 
matters  under  the  influence  of  heat. 

When  ready  it  may  be  used  as  ordinary  agar-agar  or 
gelatin,  either  for  plates  or  slant  cultures. 

Lceffler's  blood-serum  mixture.     Loeffler's  blood-serum 


SPECIAL  MEDIA.  103 

mixture  consists  of  one  part  of  neutral  meat-infusion 
bouillon  containing  1  per  cent,  of  grape-sugar,  and  three 
parts  of  blood-serum.  This  mixture  is  placed  in  test- 
tubes,  sterilized,  and  solidified  in  exactly  the  way  given 
for  blood-serum.  It  requires  for  its  solidification  a 
somewhat  higher  temperature  and  a  longer  exposure  to 
this  temperature  than  does  blood-serum  to  which  no 
bouillon  has  been  added.  (See  also  the  Councilman- 
Mai  lory  method.) 

GuarniarVs  agar-gelatin  : 


Meat  infusion 950  c.c. 

Sodium  chloride 5  grammes. 

Peptone 25-30    " 

Gelatin 40-60    " 

Agar-agar 3-4    " 

Water     .  50  c.c. 


The  point  in  the  preparation  of  this  medium  is  its 
reaction,  which  should  be  exactly  neutral. 

The  list  of  special  media  is  too  great  to  be  given  in  a 
work  of  this  size.  Their  description  must  be  seen  in 
the  original.  Those  that  have  been  given  above  will 
suffice  for  obtaining  a  clear  understanding  of  the  prin- 
ciples of  the  work. 

NOTE. — The  term  " meat-infusion"  always  implies  a 
watery  extract  of  meat  made  by  mixing  500  grammes  of 
finely  chopped  lean  meat  and  1  litre  of  water  together, 
and  allowing  them  to  stand  in  a  cool  place  for  twenty- 
four  hours.  At  the  end  of  this  time  the  fluid  portion  is 
strained  off  through  a  coarse  towel.  This  represents 
the  infusion. 


CHAPTER  VI. 


Preparation  of  the  tubes,  flasks,  etc.,  in  which  the  media  are  to  be  pre- 
served. 


WHILE  the  media  are  in  course  of  preparation  it  is 
well  to  get  the  test-tubes  and  flasks  ready  for  their 
reception,  and  it  is  essential  that  they  should  be  as  clean 
as  it  is  possible  to  make  them.  For  this  purpose  it  is 
advisable  that  both  new  tubes  and  those  which  have 
previously  been  used  should  be  boiled  for  some  time, 
about  thirty  to  forty-five  minutes,  in  a  4  to  6  per  cent, 
solution  of  common  soda  ;  it  is  not  necessary  to  be  exact 
as  to  strength,  but  it  should  not  be  weaker  than  this. 
At  the  end  of  this  time  they  are  to  be  carefully  swabbed 
out  with  a  cylindrical  bristle  brush,  preferably  one  hav- 
ing a  reed  handle  (Fig.  18),  as  those  with  wire  handles 

FIG.  18. 


Brush  for  cleaning  test-tubes. 

are  apt  to  break  through  the  bottoms  of  the  tubes.  All 
traces  of  adherent  material  should  be  carefully  removed. 
When  the  tubes  are  quite  clean  they  may  be  rinsed  in  a 
warm  solution  of  commercial  hydrochloric  acid  of  the 
strength  of  about  1  per  cent.  This  is  to  remove  the 
alkali.  They  are  then  to  be  thoroughly  rinsed  in  clear, 
running  water,  and  stood  top  down  until  the  water  has 


FILLING  THE  TUBES.  105 

drained  from  them.  When  dry  they  are  to  be  plugged 
with  raw  cotton.  The  plugging  with  the  cotton  requires 
a  little  practice  before  it  can  be  properly  done.  The 
cotton  should  be  introduced  into  the  mouths  of  the  tubes 
in  such  a  way  that  no  cracks  or  creases  exist,  but  should 
fill  them  quite  regularly  all  around.  The  plugs  should 
fit  neither  too  tightly  nor  too  loosely,  but  should  be  just 
firmly  enough  in  position  to  sustain  the  weight  of  the 
tube  into  which  it  is  placed  when  held  up  by  the  portion 
which  projects  from  and  overhangs  the  mouth  of  the 
tube.  The  tubes  thus  plugged  with  cotton  are  now  to 
be  placed  upright  in  a  wire  basket  and  heated  for  one 
hour  in  the  hot-air  sterilizer  at  a  temperature  of  about 
1 50°  C.  A  very  good  rule  for  this  process  of  sterilization 
is  to  observe  the  tubes  from  time  to  time,  and  as  soon  as 
the  cotton  has  become  a  very  light  brown  color,  not 
deeper  than  a  dark-cream  tint,  to  consider  sterilization 
complete.  The  tubes  are  then  removed  and  allowed  to 
cool  down. 

The  cotton  used  for  this  purpose  should  be  the 
ordinary  cotton  batting  of  the  shops,  and  not  absorbent 
cotton ;  the  latter  becomes  too  tightly  packed,  and  is, 
moreover,  much  too  expensive  for  this  purpose. 

Care  should  be  taken  not  to  burn  the  cotton,  other- 
wise the  tubes  will  become  coated  with  a  dark-colored, 
pyroligneous,  oily  deposit,  which  renders  them  unfit  for 
use,  and  they  will  have  to  be  cleaned  again. 

FILLING  THE  TUBES. — When  the  tubes  are  cold  they 
may  be  filled.  This  is  best  accomplished  by  the  use  of 
a  spherical  form  of  funnel,  such  as  is  shown  in  Fig.  19. 
The  liquefied  medium  is  poured  into  this  funnel,  which 
has  been  carefully  washed,  and  by  pressing  the  pinch- 
cock  with  which  the  funnel  is  provided,  the  desired 


106 


BACTERIOLOGY. 


amount  of  material  (5-10  c.c.)  may  be  allowed  to  flow 
into  the  tubes  held  under  its  opening. 

It  is  not  necessary  to  sterilize  the  funnel,  for  the 
medium  is  to  be  subjected  to  this  process  as  soon  as  it  is 
in  the  test-tubes. 

Care  should  be  taken  that  none  of  the  medium  is 
dropped  upon  the  mouth  of  the  test-tube,  otherwise  the 

FIG.  19. 


Funnel  for  filling  tubes  with  culture  media. 

cotton  plugs  become  adherent  to  it  and  are  not  only 
difficult  to  remove,  but  present  a  very  untidy  appear- 
ance, and  interfere,  indeed,  with  the  proper  manipula- 
tions. 

As  soon  as  the  tubes  have  been  filled  they  are  to  be 
sterilized  in  the  steam  sterilizer  for  fifteen  minutes  on 


FILLING  THE  TUBES.  107 

each  of  three  successive  days.  During  the  intervening 
days  they  may  be  kept  at  the  ordinary  room  tempera- 
ture. 

When  sterilization  is  complete,  and  the  medium  in 
the  tubes  is  still  liquid,  some  of  them  may  be  placed  in 
a  slanting  position,  at  an  angle  of  about  ten  degrees  with 
the  surface  on  which  they  rest,  and  the  medium  allowed 
to  solidify  in  this  position.  These  are  for  the  so-called 
slant  cultures.  The  balance  may  solidify  in  the  erect 
position ;  these  serve  for  the  plate  cultures. 

For  Esrnarch  tubes  not  more  than  5  c.c.  of  material 
should  be  placed  in  each  tube,  as  more  than  this  renders 
the  rolling  difficult  and  irregular. 


CHAPTER   VII. 

Technique  of  making  plates— Esmarch  tubes,  Petri  plates,  etc. 

PLATES. — The  plate  method  can  be  practised  with 
both  agar-agar  and  gelatin.  It  cannot  be  practised 
with  blood-serum,  because  the  serum,  when  once  solidi- 
fied, cannot  be  again  liquified. 

Plates  are  usually  referred  to  as  "  a  set."  This  term 
implies  three  individual  plates,  each  representing  the 
mixture  of  organisms  in  a  higher  state  of  dilution. 
The  first  plate  is  known  usually  as  "the  original,"  or 
"plate  1,"  the  first  dilution  from  this  as  "plate  2,"  and 
the  second  as  "plate  3." 

In  the  preparation  of  a  set  of  plates  the  following 
are  the  steps  to  be  observed  : 

Three  tubes,  each  containing  from  7  to  9  c.c.  of  gela- 
tin or  agar-agar,  are  placed  in  the  warm  water  bath 
until  the  medium  has  become  liquid.  If  agar-agar  is 
employed,  this  is  accomplished  at  the  boiling-point  of 
water ;  if  gelatin  is  used,  a  much  lower  temperature 
suffices  (85°-40°  C.).  When  liquefaction  is  complete 
the  temperature  of  the  water,  in  the  case  of  agar-agar, 
must  be  reduced  to  41°-42°  C.,  at  which  temperature 
the  agar-agar  remains  liquid,  and  the  organisms  may  be 
introduced  into  it  without  fear  of  destroying  their  vital- 
ity. The  medium  being  now  liquid  and  of  the  proper 
temperature,  a  very  small  portion  of  the  mixture  of 
organisms  to  be  studied  is  taken  up  with  a  sterilized, 


TECHNIQUE  OF  MAKING  PLATES.  .  109 

looped  platinum  wire,  Fig.  20.  This  is  nothing  more 
than  a  piece  of  platinum  wire  of  about  5  em.  long, 
twisted  into  a  small  loop  at  one  end  and  fused  into  a 
bit  of  glass  rod,  which  acts  as  a  handle,  at  the  other 
extremity.  This  loop  is  one  of  the  most  useful  of 
bacteriological  instruments,  as  there  is  hardly  a  manipu- 
lation in  the  work  into  which  it  does  not  enter.  Under 
no  conditions  is  it  to  be  employed  without  having  been 


FIG.  20. 
a 


6 
Looped  and  straight  platinum  wires  in  glass  handles. 

passed  through  the  gas-flame  until  quite  hot ;  this  is  for 
the  purpose  of  sterilization.  One  should  form  a  habit 
of  never  taking  up  one  of  these  platinum-wire  needles, 
as  they  are  called,  for  they  are  both  looped  and  curved 
or  straight  (Fig.  20,  6),  without  passing  it  through  the 
flame,  and  the  sooner  the  beginner  learns  to  do  this  as  a 
matter  of  reflex,  the  sooner  does  he  rid  himself  of  one 
of  the  possible  sources  of  error  in  his  work.  It  must 
be  remembered,  though,  that  it  should  not  be  used  when 
hot,  otherwise  the  organisms  taken  upon  it  are  killed 
by  the  high  temperature ;  after  sterilization  in  the  flame 
one  waits  for  a  few  seconds  until  it  is  cool  before  using. 
The  bit  of  material  under  consideration  is  transferred 
with  the  sterilized  loop  into  tube  No.  1,  "the  original," 
where  it  is  carefully  disintegrated  by  gently  rubbing  it 
against  the  sides  of  the  tube.  The  more  carefully  this 

6 


HO  BACTERIOLOGY. 

is  done  the  more  homogenous  will  be  the  distribution 
of  the  organisms  and  the  better  the  results.  The  loop 
is  then  again  sterilized,  and  three  of  its  loopfuls  are 
passed,  without  touching  the  sides  of  the  tube,  from  u  the 
original "  into  tube  No.  2,  where  they  are  carefully 
mixed.  Again  the  loop  is  sterilized,  and  again  three 
dips  are  made  from  tube  2  into  tube  3.  This  completes 
the  dilution.  The  loop  is  now  sterilized  before  laying 
it  aside. 

FIG.  21. 


Levelling  tripod  with  glass  chamber  for  plates. 

During  this  manipulation,  which  must  be  done 
quickly  if  agar-agar  be  employed,  the  temperature  of 
the  water  in  the  bath  in  which  the  tubes  stand  should 
never  get  lower  than  39°  C.,  and  never  higher  than 
43°  C.  If  it  falls  too  low,  below  38°  C.,  the  agar-agar 
gelatinizes,  and  can  only  be  redissolved  by  a  tempera- 
ture that  would  be  destructive  to  the  organisms  which 
may  have  been  introduced  into  the  tubes.  This  is  not 
of  so  much  moment  with  gelatin,  as  it  may  readily  be 


THE  COOLING  PLATES.  HI 

redissolved  at  a  temperature  not  detrimental  to  the 
organisms  with  which  the  tubes  may  have  been  inocu- 
lated. 

THE  COOLING-STAGE  AND  LEVELLING  TRIPOD. — 
While  the  medium  of  which  the  plates  are  to  be  made 
is  melting,  it  is  well  to  arrange  the  cooling-stage  (Fig. 
21)  upon  which  the  gelatin  or  agar-agar  is  to  be  subse- 
quently solidified. 

This  stage  consists  of  a  glass  dish  filled  with  ice- 
water  and  covered  with  a  ground-glass  plate,  which  in 
turn  has  a  dome-shaped  cover.  The  dish  rests  upon  a 
tripod  which  can  be  brought  to  an  exact  level,  as  indi- 
cated by  the  spirit-level,  by  raising  or  lowering  its  legs 
by  means  of  thumb-screws,  with  which  they  are  pro- 
vided. Three  stages  are  usually  employed.  When  ready 
for  use  they  should  be  exactly  level. 

FIG.  22. 


Russia  iron  box  for  holding  plates,  etc.,  during  sterilization  in  dry  heat. 


THE  GLASS  PLATES. — On  each  of  the  stages  is  to  be 
placed  a  glass  plate  upon  which  the  liquefied  gelatin  or 
agar-agar  is  to  be  poured  and  allowed  to  solidify.  It 
is,  therefore,  necessary  that  the  plates  should  not  only 
be  sterile  when  placed  upon  the  stages,  but  should  be 
carefully  protected  by  a  cover  against  dust  and  bacteria 
from  outside  sources  during  manipulation. 


112  BACTERIOLOGY. 

A  number  of  plates  at  a  time  are  usually  sterilized  in 
the  dry  sterilizer  at  a  temperature  of  150°  to  180°  C. 
for  one  hour.  During  sterilization  and  until  used  they 
are  retained  in  an  iron  box  (Fig.  22),  which  is  especially 
designed  for  the  purpose. 

They  should  never  be  placed  upon  the  stage  until 
cold  ;  otherwise  they  crack. 

When  the  plates  which  have  been  placed  upon  the 
stages  are  quite  cold,  the  melted  gelatin  or  agar-agar  in 
the  tubes  which  represent  the  three  dilutions  should  be 
poured  upon  them,  each  tube  being  emptied  upon  a 
separate  plate.  If  the  medium  is  quite  fluid  it  spreads 
over  the  surface  of  the  plates  in  a  thin,  even  layer. 
Sometimes  it  may  be  more  evenly  spread  as  it  flows 
from  the  tube  by  the  aid  of  a  sterilized  glass  rod. 

FIG.  23. 


Glass  benches  for  supporting  plates. 

As  the  contents  of  each  tube  are  emptied  upon  a  plate 
the  cover  of  the  cooling-stage  is  quickly  replaced  and 
the  plate  allowed  to  stand  until  the  gelatin  or  agar-agar 
is  quite  solid.  This  takes  longer  with  gelatin  than  with 
agar.  When  quite  solid  they  are  placed  upon  little 
glass  benches  (Fig.  23),  and  each  bench  is  labelled  with 
the  number  of  the  plate  in  the  series  of  dilutions.  The 
benches,  with  the  plates  upon  them,  are  then  piled  one 
above  the  other  in  a  glass  dish,  the  so-called  u  culture- 
dish,"  in  which  the  plates  are  to  be  kept  during  the 


CULTURE-DISH.  113 

growth  of  the  bacteria.  The  benches  are  sterilized 
before  using,  in  the  way  given  for  the  plates. 

CULTURE-DISH. — This  dish,  which  is  about  22  cm. 
in  diameter  and  has  vertical  sides  of  about  6  cm.  in 
height,  is  provided  with  a  cover  of  exactly  the  same 
design,  but  of  a  little  larger  diameter.  This  cover, 
when  placed  upon  the  dish  containing  the  plates,  fits 
over  it  and  prevents  the  access  of  dust.  Prior  to  using, 
the  dish  and  cover  should  have  been  disinfected  for  one- 
half  hour  with  1  : 1000  sublimate,  and  then  all  the  sub- 
limate solution  allowed  to  drain  from  it. 

In  the  bottom  of  this  dish  is  sometimes  placed  a 
disk  of  sterilized  filter-paper  moistened  with  sterilized 
water,  which  serves  to  prevent  the  drying  of  the  plates. 
This,  however,  is  not  necessary. 

If  agar-agar  be  employed,  the  dish  and  its  contents 
may  be  placed  at  a  temperature  of  37°-38°  C.;  if 
gelatin,  the  temperature  at  which  the  plates  are  now  to 
be  kept  should  not  be  over  22°  C.,  otherwise  the  gela- 
tin becomes  liquefied  and  the  plates  are  rendered  useless. 

When  development  has  occurred,  the  object  of  the 
dilution  will  easily  be  seen,  and  the  different  species  of 
bacteria  in  the  mixture  will  be  recognized  by  differences 
in  the  character  of  the  colonies  growing  from  them. 

This,  in  short,  is  the  plate  method  of  Koch  for  the 
separation  of  the  individual  species  contained  in  a  mix- 
ture of  bacteria.  Many  modifications  of  this  method 
exist  ;~all,  however,  are  based  upon  the  same  principles. 
The  modifications  have  for  their  object  the  accomplish- 
ment of  the  same  end,  but  with  a  smaller  armamenta- 
rium of  apparatus,  and  in  general  the  one  or  the  other 
of  these  modifications  has  entirely  supplanted  the  origi- 
nal plate  method  as  practised  and  recommended  by  Koch. 


1 14  BACTEEIOLOO  Y. 

PETRI'S  MODIFICATION  OF  THE  PLATE  METHOD. — 
The  modification  which  approaches  nearest  to  the  original 
method,  and  at  the  same  time  lessens  very  materially  the 
number  of  steps  in  the  process,  is  that  suggested  by  Petri. 
It  consists  in  substituting  for  the  plates  small,  round, 
double  glass  dishes,  which  have  about  the  same  surface- 
area  as  the  plates.  The  liquid  medium  may  be  poured 
directly  into  these  little  dishes  without  their  being  exactly 
level.  Each  dish  acts  as  a  plate.  Their  covers  are  then 
to  be  replaced,  and  they  are  set  aside  for  observation. 
In  all  other  respects  the  steps  are  the  same  as  those  given 
for  Koch's  original  method.  Petri's  dishes  are  flat,  double 
dishes  of  glass  (Fig.  24).  They  are  of  about  8  cm.  in 

FIG.  24. 


Petri  double  dish,  now  generally  used  Instead  of  plates. 

diameter  and  about  1.5  to  2  cm.  in  height,  the  walls 
being  vertical.  They  may  readily  be  sterilized  either 
by  the  hot-air  or  steam  methods  of  sterilization.  They 
are  very  useful  for  this  work,  as  they  do  away  with  the 
necessity  for  the  cooling-stage  and  levelling  tripods, 
though  in  warm  weather  the  cooling-stage  may  be  used 
to  hasten  the  solidification  of  gelatin.  A  cooling-stage 
of  very  convenient  design  for  use  with  these  dishes  con- 
sists of  a  closed,  flat  metal  box,  either  of  copper  or 
block  tin,  and  either  round  or  square  in  shape,  so 
arranged  that  it  can  be  filled  with  cold  water,  or  that 
cold  water  can  constantly  be  passed  through  it  by 


ESMARCWS  TUBES.  115 

means  of  a  rubber  connection  with  a  spigot.  The 
inlet  for  the  water  should  be  just  above  the  bottom 
of  the  box,  and  the  outlet  just  beneath  the  top  and 
slightly  turned  upward  and  then  downward,  so  as  to 
insure  the  complete  filling  of  the  space  with  water.  The 
box  should  be  sufficiently  strong  to  resist  the  pressure  of 
the  water.  A  convenient  size  is  from  20  to  25  cm.  in 
diameter,  and  of  about  1.5  to  2  cm.  high.  It  is  simple 
in  construction,  and  can  be  made  by  any  copper  spinner. 
An  idea  of  its  construction  is  given  in  Fig.  25. 

FIG.  25. 


Metal  cooling-stage. 

When  gelatin  or  agar-agar  is  to  be  cooled  it  is  only 
necessary  to  place  the  dishes  containing  it  on  top  of  this 
box  and  start  cold  water  circulating  through  it. 

ESMARCH'S  TUBES. — The  modification  of  Koch's 
method  which  insures  the  greatest  security  from  con- 
tamination by  outside  organisms  and  requires  the  small- 
est supply  of  apparatus  is  that  suggested  by  v.  Esmarch. 
It  differs  from  the  other  methods  thus  :  The  dilutions 
having  been  prepared  in  tubes  containing  a  smaller 
amount  of  medium  than  usual — as  a  rule  not  more  than 
5  to  6  c.c. — are,  instead  of  being  poured  out  upon  plates 
or  into  dishes,  spread  over  the  inner  surface  of  the  tube 


116  BACTERIOLOGY. 

containing  them,  and  without  removing  the  cotton  plugs 
are  caused  to  solidify  in  this  position.  The  tubes  then 
present  a  thin  cylindrical  lining  of  gelatin  or  agar-agar, 
upon  which  the  colonies  develop.  In  all  other  respects 
the  conditions  for  the  growth  of  the  organisms  are  the 
same  as  in  flat  plates. 

Esmarch  directs  that  after  completion  of  the  dilutions 
the  tops  of  the  cotton  plugs  in  the  tubes  should  be  cut 
off  flush  with  the  mouth  of  the  test-tube  and  a  steril- 
ized rubber  cap  be  placed  over  this.  They  are  then  to 
be  held  in  the  horizontal  position  and  twisted  between 
the  fingers  upon  their  long  axis  under  ice-water.  The 
gelatin  becomes  solidified  thereby  and  adheres  to  the 
sides  of  the  tube.  When  the  gelatin  is  quite  hard  the 
tubes  are  removed  from  the  water,  wiped  dry,  the  rub- 
ber caps  removed,  and  they  are  set  aside  for  observation. 

For  some  time  past  we  have  deviated  from  the  direc- 
tion given  by  v.  Esmarch  for  this  part  of  his  method, 
and  instead  of  rolling  the  tubes  under  ice-water,  we  roll 
them  upon  a  block  of  ice  (Fig.  26),  after  the  method 
devised  by  Booker  in  the  Pathological  Laboratory  of 
the  Johns  Hopkins  University  in  1887.  In  this  method 
a  small  block  of  ice  only  is  needed.  It  is  arranged 
nearly  level,  and  is  held  in  position  by  being  placed  in 
a  dish  upon  a  te^el.  A  horizontal  groove  is  melted  in 
the  surface  of  the  ice  with  a  test-tube  full  of  hot  water. 
The  tubes  to  be  rolled  are  then  held  in  an  almost,  not 
quite,  horizontal  position  and  twisted  between  the  fingers 
until  the  sides  are  moistened  by  the  contents  to  within 
about  1  cm.  of  the  cotton  plug,  care  being  taken  that 
the  gelatin  does  not  touch  the  cotton ;  otherwise  the  latter 
becomes  adherent  to  the  sides  of  the  tube  and  is  difficult 
to  remove.  The  tube  is  then  placed  in  the  groove  in 


ESM 'ARCH'S  TUBES.  117 

the  ice  and  rolled,  neither  rubber  cap  nor  cutting  off  of 
the  cotton  plug  being  necessary. 


FIG.  26. 


Demonstrating  Booker's  method  of  rolling  Esmarch  tubes  on  a  block  of  ice. 

The  advantages  of  this  process  over  that  followed  by 
v.  Esmarch  are  that  it  requires  less  time,  is  cleaner,  no 
rubber  caps  are  needed,  the  rolled  tubes  are  more  regu- 
lar, and  the  gelatin  does  not  touch  the  cotton  plug,  as 
is  always  the  case  in  the  tubes  rolled  under  water,  be- 
cause of  the  impossibility  of  holding  them  steady  at  one 
level. 

There  is  an  impression  that  Esmarch  tubes  are  not 
a  success  when  made  from  ordinary  nutrient  agar-agar 
because  of  the  tendency  of  this  medium  to  collapse  and 
fall  into  the  bottom  of  the  tube.  This  slipping  down 
of  the  agar-agar  is  due  to  the  water  that  is  squeezed 
from  it  during  solidification  getting  between  the  medium 
and  the  walls  of  the  tube.  This  can  easily  be  overcome 
by  allowing  the  rolled  tubes  to  remain  in  nearly  a  hori- 
zontal position,  the  cotton  end  of  the  tube  about  1.5  to 
2  cm.  higher  than  the  bottom  of  the  tube,  for  twenty- 

6* 


118  BACTERIOLOGY. 

four  hours  after  rolling  them.  Daring  this  time  the 
edge  of  the  agar-agar  nearest  the  cotton  plug  becomes 
dried  and  adherent  to  the  walls  of  the  tube,  while  the 
water  collects  at  the  most  dependent  point,  i.  e.,  the  bot- 
tom of  the  tube.  After  this  they  may  be  retained  in 
the  upright  position  without  fear  of  the  agar-agar  slip- 
ping down.  We  have  followed  this  process  for  several 
years  with  entire  satisfaction.1 

In  all  these  processes,  if  the  dilutions  of  the  number 
of  organisms  have  been  properly  conducted,  the  results 
will  be  the  same.  The  original  plate  or  tube,  as  a  rule, 
will  be  of  no  use  because  of  the  great  number  of  col- 
onies in  it.  Plate  or  tube  No.  2  may  be  of  service,  but 
plate  or  tube  3  will  usually  contain  the  organisms  in 
such  small  numbers  that  the  colonies  originating  from 
them  will  have  nothing  to  prevent  their  characteristic 
development. 

For  reasons  of  economy,  the  "original,"  tube  1,  is 
sometimes  substituted  by  a  tube  containing  normal  salt 
solution  (0.6  to  0.7  per  cent,  of  sodium  chloride  in 
water),  which  is  thrown  aside  as  soon  as  the  dilutions 
are  completed,  and  only  plates  or  tubes  '2  and  3  are  made. 

Another  method  for  the  separation  of  bacteria  and 
their  isolation  as  single  colonies  consists  in  the  making 
of  dilutions  upon  the  surface  of  solid  media,  such  as 
potato,  coagulated  blood-serum,  agar-agar,  and  gelatin. 
For  the  performance  of  this  method  one  selects  a  num- 
ber of  tubes  containing  the  medium  to  be  employed  in 
a  slanting  position.  With  the  platinum  needle  a  bit  of 
the  substance  to  be  studied  is  smeared  upon  tube  No.  1 ; 


1  The  impression  that  agar-agar  is  not  suitable  for  roll  tubes  was  shown  to 
be  erroneous,  and  the  above  method  was  developed  in  the  Pathological  Lab- 
oratory of  the  Johns  Hopkins  University. 


ESMARCH'S  TUBES.  119 

without  sterilizing  the  needle  it  is  passed  thoroughly 
over  the  surface  of  the  medium  in  tubes  2,  3,  4,  etc., 
etc.,  in  succession.  When  development  has  occurred 
essentially  the  same  conditions  as  regards  separation  of 
the  colonies  will  be  found  as  is  the  case  when  plates  are 
poured.  If  a  slanted  medium  be  employed,  about  the 
most  dependent  angle  of  which  water  of  condensation 
has  accumulated,  as  blood-serum,  agar-agar,  and  potato, 
the  dilutions  may  be  made  in  this  fluid,  and  this  is  then 
to  be  carefully  smeared  over  the  solid  surface  of  the  me- 
dium. The  tubes  thus  treated  should  be  kept  in  an  up- 
right position  to  prevent  the  fluid  from  flowing  over  the 
surface.  When  sufficiently  developed,  single  colonies 
may  be  isolated  from  tubes  prepared  in  this  manner 
with  comparative  ease.  (See  also  method  for  the  isola- 
tion of  b.  diphtherice  on  blood-serum.) 


CHAPTER  VIII. 

The  incubating  oven— Gas-pressure   regulator  —  Thermo-regulator  —  The 
safety  burner  employed  in  heating  the  incubator. 

THE  INCUBATOR. — When  the  plates  have  been  made 
it  must  be  borne  in  mind  that  for  the  development  of 
certain  forms  of  bacteria  a  higher  temperature  is  neces- 
sary than  for  the  growth  of  others.  The  pathogenic 
or  disease-producing  organisms  all  grow  more  luxuri- 
antly at  the  temperature  of  the  human  body  (37.5°  C.) 
than  at  lower  temperatures ;  whereas,  with  the  ordinary 
saphrophytic  forms  almost  any  temperature  between 
18°  C.  and  that  of  the  body  is  suitable.  It  therefore 
becomes  necessary  to  provide  some  place  in  which  a 
constant  temperature  favorable  to  the  growth  of  the 
pathogenic  organisms  can  be  maintained.  For  this  pur- 
pose there  have  been  devised  a  number  of  different 
forms  of  apparatus.  Fundamentally  they  are  all  based 
upon  the  same  principles,  however,  and  a  general  de- 
scription of  the  essential  points  involved  in  their  con- 
struction will  be  all  that  is  needed  here. 

This  apparatus  has  the  names  thermostat,  incubator, 
and  brooding  oven.  It  is  a  copper  chamber  (Fig.  27) 
with  double  walls,  the  space  between  which  is  filled 
with  water.  The  incubating  chamber  may  be  opened 
or  closed  by  a  closely  fitting  double  door,  inside  of  which 
is  usually  a  false  door  of  glass  through  which  the  con- 
tents of  the  chamber  may  be  inspected  without  actually 
opening  it.  The  whole  apparatus  is  encased  in  either 


THE  INCUBATOR. 


121 


asbestos  boards  or  thick  felt,  to  prevent  radiation  of  heat 
and  consequent  fluctuations  in  temperature.  In  the  top 
of  the  chamber  is  a  small  opening  through  which  a 


FIG.  27. 


Incubator  used  in  bacteriological  work. 

thermometer  projects  into  its  interior.  At  either  corner, 
leading  into  the  space  containing  the  water,  are  other 
openings  for  the  reception  of  another  thermometer  and 
a  thermo-regulator,  and  for  refilling  the  apparatus  as 
the  water  evaporates.  On  the  side  is  a  water-gauge  for 


122 


BACTERIOLOGY. 


showing  the  level  of  the  water  between  the  walls.  The 
object  of  the  water  chamber,  which  is  formed  by  the 
double  wall  arrangement,  is  to  insure,  by  means  of  the 
warmed  water,  an  equable  temperature  at  all  parts  of 
the  apparatus — at  the  top  as  well  as  at  the  sides,  back, 
and  bottom,  and  the  apparatus  should  be  kept  filled 
with  water,  otherwise  the  purpose  for  which  it  is  con- 
structed will  not  be  accomplished.  When  the  chamber 
between  the  walls  is  filled  with  water  heat  is  supplied 
from  a  gas-flame  placed  beneath  it. 


FIG.  28. 


Koch's  safety  burner. 


The  burner  employed  in  heating  the  incubator  was 
originally  devised  by  Koch,  and  is  known  as  u  Koch's 
safety  burner  "  (Fig.  28).  It  is  a  Bunsen  burner  pro- 
vided with  an  arrangement  for  automatically  turning 


THERMO-REG  ULA  TORS.  123 

off  the  gas  supply  and  thus  preventing  accidents  should 
the  flame  become  extinguished  at  a  time  when  no  one 
was  near.  The  gas-cock  by  which  the  gas  is  turned  on 
and  off  is  provided  with  a  long  arm  which  is  weighted 
and  which,  when  the  gas  is  turned  on  and  burning,  rests 
upon  the  side  of  a  revolving,  horizontal  disc  that  is  con- 
nected with  the  free  ends  of  two  metal  spirals  which  are 
fixed  by  their  other  ends  in  opposite  directions  on  either 
side  of  the  flame  and  heated  by  it.  If  by  draughts  or 
any  other  accident  the  flame  becomes  extinguished  the 
metal  spirals  cool,  and  in  cooling  contract,  twist  the 
horizontal  disc  in  the  opposite  direction  and  allow  the 
weighted  arm  of  the  gas-cock  to  fall.  By  its  falling  the 
gas  supply  is  turned  off. 

THERMO-REGULATORS. — The  regulation  and  mainte- 
nance of  the  proper  temperature  within  the  incubator 
is  accomplished  by  the  employment  of  an  automatic 
thermo- regulator. 

The  common  form  of  thermo-regulator  used  for  this 
purpose  is  constructed  upon  principles  involving  the 
expansion  and  contraction  of  fluid  substances  under  the 
influence  of  heat  and  cold.  By  means  of  this  expan- 
sion and  contraction  the  amount  of  gas  passing  from 
the  source  of  supply  to  the  burner  may  be  either  dimin- 
ished or  increased  as  the  temperature  of  the  substance 
in  which  the  regulator  is  placed  either  rises  or  falls. 

The  simplest  form  of  thermo-regulator  which  serves 
to  illustrate  the  principles  involved  is  seen  in  Fig.  29. 

It  consists  of  a  glass  cylinder  e,  having  a  communi- 
cating branch  tube  6,  and  rubber  stopper  /,  through 
which  projects  the  bent  tube  a.  The  tube  a  is  ground 
to  a  slanting  point  at  the  extremity  which  projects  into 


124 


BACTERIOLOGY. 


the  tube  e,  and  is  provided  a  short  distance  above  this 
point  with  a  capillary  opening  g,  in  one  of  its  sides. 


FIG.  29. 


Mercurial  thermo-regulator. 

When  ready  for  use  the  cylinder  e  is  filled  with  mercury 
up  to  about  the  level  shown  in  the  figure.  It  is  then 
allowed  to  stand,  or  is  suspended,  in  the  bath  the  tem- 
perature of  which  it  is  to  regulate.  The  rubber  tubing 
coming  from  the  gas  supply  is  attached  to  the  outer  end 
of  the  glass  tube  a,  and  the  tube  going  to  the  burner  is 
slipped  over  the  branch  tube  b.  The  gas  is  turned  on  and 
the  burner  lighted  and  placed  under  the  bath.  The  gas 


THERMO-REG  ULATORS.  125 

DOW  streams  through  the  tube  a  into  the  cylinder  e  and 
out  at  b  to  the  burner,  but  as  the  temperature  of  the 
bath  rises,  the  mercury  contained  in  the  cylinder  e,  under 
the  influence  of  the  elevation  of  temperature  begins  to 
expand,  and,  as  a  continuous  rise  in  temperature  pro- 
ceeds, the  expansion  of  the  fluid  accompanies  it  and 
gradually  closes  the  slanting  opening  h  of  tube  a.  In 
this  way  the  supply  of  gas  becomes  diminished  and 
the  rise  in  temperature  of  the  bath  will  be  less  rapid, 
until  finally  the  opening  at  h  will  be  closed  entirely, 
when  the  supply  of  gas  to  the  burner  will  now  be 
limited  to  that  passing  through  the  capillary  opening 
g.  This  is  not  sufficient  to  maintain  the  highest  tem- 
perature reached,  and  as  cooling  begins  a  gradual  contrac- 
tion of  the  mercury  occurs  until  there  is  again  an  outflow 
of  gas  from  the  opening  A,  when  again  the  temperature 
rises.  This  contraction  and  expansion  of  the  mercury 
in  the  regulator  continues  until  eventually  a  point  is 
reached  at  which  its  position  in  the  cylinder  e  allows 
of  the  passage  of  just  enough  gas  from  the  opening  h  to 
maintain  a  constant  temperature ;  and,  therefore,  a  con- 
stant degree  of  expansion  of  the  mercury  in  the  tube  e. 
This,  in  short,  is  the  principle  on  which  thermo-regu- 
lators  are  constructed,  but  it  must  be  borne  in  mind  that 
a  great  deal  of  detail  exists  in  the  construction  of  an 
accurate  instrument.  The  number  of  different  forms  of 
this  apparatus  is  comparatively  large,  and  each  form  has 
its  special  merits. 

The  value,  that  is  the  delicacy,  of  the  thermo- 
regulator  depends  upon  a  number  of  factors,  all  of 
which  it  would  be  useless  to  introduce  into  a  book  of 
this  kind,  but  in  general  it  may  be  said  that  the  essen- 
tial points  to  be  observed  in  selecting  a  thermo-regulator 


126 


BACTERIOLOGY. 


depend  in  the  main  upon  the  temperatures  to  which  it 
is  to  be  applied.  For  low  temperatures,  regulators  con- 
taining such  fluids  as  ether,  alcohol,  and  calcium  chloride 
solution,  which  expand  and  contract  rapidly  and  regu- 
larly under  slight  variations  in  temperature,  are  com- 
monly employed  ;  whereas  for  temperatures  approaching 
the  boiling-point  of  water  mercury  is  most  frequently 
used. 

The  temperature  of  the  incubator  is  to  be  regulated, 
then,  by  the  use  of  some  such  form  of  apparatus  as  that 
just  described.  It  should  be  of  sufficient  delicacy  to 
prevent  a  fluctuation  of  more  than  0.2°  C.  in  the  tem- 
perature of  the  air  within  the  chamber  of  the  apparatus. 

FIG.  30. 


Moitessier's  gas-pressure  regulator. 

GAS-PRESSURE  REGULATORS. — A  gas-pressure  regu- 
lator is  not  rarely  intervened  between  the  gas  supply 


GAS-PRESS  URE  REG  ULA  TORS.  127 

and  the  therm o- regulator.  This  apparatus  has  for  its 
object  the  maintenance  of  a  constant  pressure  of  the 
gas  going  to  the  therrao-regulator.  There  are  several 
instruments  of  this  form  in  use,  but  they  do  not  accom- 
plish the  object  for  which  they  are  designed. 

The  instrument  most  commonly  employed,  the  appa- 
ratus of  Moitessier  (Fig.  30),  is  based  on  somewhat  the 
same  principles  as  the  large  regulators  seen  at  the 
manufactories  of  illuminating  gas.  Such  apparatus  act 
very  well  when  employed  on  the  large  scale,  as  one  sees 
them  at  the  gas-works ;  but  when  applied  to  the  limited 
and  sudden  fluctuations  seen  in  the  gas  coming  from  an 
ordinary  gas-cock,  are  practically  useless.  They  are  too 
gross  in  their  construction,  and  act  only  under  compara- 
tively great  and  gradual  fluctuations  in  pressure.  If  a 
good  form  of  thermo-regulator  be  employed  there  is  no 
necessity  for  the  use  of  any  of  the  forms  of  pressure- 
regulators  thus  far  introduced. 


CHAPTER  IX. 

The  study  of  colonies— Their  naked-eye  peculiarities  and  their  appearance 
under  different  conditions— Differences  in  the  structure  of  colonies  from 
different  species  of  bacteria— Stab  cultures— Slant  cultures. 

THE  plates  upon  agar-agar  which  have  been  prepared 
from  a  mixture  of  organisms  and  have  been  placed  in 
the  incubator,  and  those  of  gelatin  which  have  been 
maintained  at  the  ordinary  temperature  of  the  room,  are 
usually  ready  for  examination  after  twenty-four  to 
forty-eight  hours.  They  will  be  found  to  be  marked, 
here  and  there,  by  small  points  or  little  islands  of  more 
or  less  opaque  appearance.  In  some  instances  these 
will  be  so  transparent  that  it  is  with  difficulty  one  can 
see  them  with  the  naked  eye.  Again,  they  may  be  of 
a  dense  opaque  appearance,  at  one  time  sharply  circum- 
scribed and  round,  again  irregular  in  their  outline ; 
here  a  point  will  present  one  color,  there  perhaps 
another.  On  gelatin  some  of  the  points  will  be  seen  to 
be  lying  on  the  surface  of  the  medium,  others  will  have 
sunk  into  little  depressions,  while  at  still  other  points 
the  clear  gelatin  will  be  marked  by  more  or  less  saucer- 
shape  pits  containing  opaque  fluid. 

Place  the  plate  containing  these  points  upon  the  stage 
of  the  microscope  and  examine  them  with  the  lowest 
power  objective,  and  again  differences  will  be  observed. 
Some  of  these  minute  points  will  be  finely  granular, 
others  coarsely  so;  some  will  present  a  radiated  appear- 
ance, while  a  neighbor  may  be  concentrically  arranged ; 


THE  STUDY  OF  COLONIES.  129 

here  nothing  particularly  characteristic  will  present, 
there  the  point  may  resolve  itself  into  a  little  mass  hav- 
ing somewhat  the  appearance  of  a  very  small  pellicle  of 
raw  cotton.  All  these  differences,  and  many  more,  aid 
us  in  saying  that  these  little  points  must  be  different 
in  their  nature.  With  a  pointed  platinum  needle  take 
up  a  bit  of  one  of  these  little  islands,  prepare  it  for 
microscopic  examination  (see  chapter  on  stained  cover- 
slip  preparations),  and  examine  it  under  the  high  power 
oil-immersion  objective,  under  access  of  the  greatest 
amount  of  light  afforded  by  the  illuminator  of  the 
microscope.  The  preparation  will  be  seen  to  be  made 
up  entirely  of  bodies  of  the  same  shape ;  they  will  all 
be  spheres,  or  ovals,  or  rods,  but  not  a  mixture  of  these 
forms,  if  proper  care  in  the  manipulation  has  been 
taken.  Examine  in  the  same  way  a  neighboring  spot 
which  possesses  different  naked-eye  appearances,  and  it 
will  often  be  found  to  consist  of  bodies  of  an  entirely  dif- 
ferent appearance  from  those  seen  in  the  first  preparatiou. 

These  spots  or  islands  on  the  surface  of  the  plates  are 
colonies  of  bacteria,  differing  severally,  not  only  in  out- 
ward appearances,  the  one  from  the  other,  but,  as  our 
cover-slip  preparations  show,  in  the  morphological  char- 
acteristics of  the  individual  organisms  composing  them. 
If  from  one  of  these  colonies  a  second  set  of  plates  be 
prepared,  the  peculiarities  which  were  at  first  observed 
in  this  colony  will  be  reproduced  in  all  of  the  new  set 
of  colonies  which  develop ;  each  will  be  found  to  con- 
sist of  the  same  organisms  as  the  colony  from  which  the 
plates  were  made.  lu  other  words,  these  peculiarities 
are  constant  under  constant  conditions. 

With  all  organisms  differences  in  the  appearance  of 
the  colonies  depending  upon  their  location  in  the  medium 


130  BACTERIOLOGY. 

can  usually  be  detected.  When  deep  down  in  the 
medium,  owing  to  surrounding  pressure,  they  are  quite 
round,  oval,  or  lozenge-shape ;  whereas,  when  they  are 
on  the  surface  of  the  gelatin  or  agar,  they  may  take 
quite  a  dhTerent  form.  This  is  purely  a  mechanical 
effect  due  to  the  pressure  of,  or  resistance  offered  by,  the 
medium  surrounding  them,  and  is  always  to  be  borne  in 
mind,  otherwise  errors  are  apt  to  arise. 

PURE  CULTURES. — If  from  one  of  these  small  colonies 
a  bit  be  taken  upon  the  point  of  a  sterilized  platinum 
needle  and  introduced  into  a  tube  of  sterilized  gelatin 
or  agar-agar,  the  growth  that  results  will  be  what  is 
known  as  a  "  pure  culture/'  the  condition  to  which  all 
organisms  must  be  brought  before  a  systematic  study  of 
their  many  peculiarities  is  begun.  Sometimes  several 
series  of  plates  are  necessary  before  the  organism  can  be 
obtained  pure,  but  by  patiently  following  this  plan  the 
results  will  ultimately  be  satisfactory. 

TEST-TUBE  CULTURES;  STAB  CULTURES;  SMEAR 
CULTURES. — After  separating  the  organisms,  the  one 
from  the  other  by  the  plate  method  just  described,  they 
must  be  isolated  from  the  plates  as  pure  stab  or  smear 
cultures. 

This  is  done  in  the  following  way  :  Decide  upon  the 
colony  from  which  the  pure  culture  is  to  be  made. 
Select  preferably  a  small  colony  and  one  as  widely  sep- 
arated from  other  colonies  as  possible.  Sterilize  in  the 
gas-flame  a  straight  platinum-wire  needle.  The  glass 
handle  of  the  needle  should  be  drawn  through  the  flame 
as  well  as  the  needle  itself,  otherwise  contamination 
from  this  source  may  occur.  When  it  is  cool,  which  is 
in  five  or  ten  seconds,  take  up  carefully  a  portion  of  the 
colony.  Guard  against  touching  anything  but  the  colony. 


TEST-TUBE  CULTURES,  ETC. 

If  during  manipulation  the  needle  touches  anything 
else  whatever  than  the  colony  from  which  the  culture  is 
to  be  made,  it  must  be  sterilized  again.  This  holds  not 
only  for  the  time  before  touching  the  colony,  but  also 
during  its  passage  into  the  test-tube  from  the  colony, 
otherwise  there  is  no  guarantee  that  the  growth  resulting 
from  the  inoculation  of  this  bit  of  colony  into  a  fresh 
sterile  medium  will  be  pure. 

In  the  meantime  have  in  the  other  hand  a  test-tube 
of  sterile  medium  :  gelatin,  agar-agar,  or  potato.  This 
tube  is  held  across  the  palm  of  the  hand  in  an  almost 
horizontal  position  with  its  mouth  pointing  out  between 
the  thumb  and  index  finger  and  its  contents  toward  the 
body  of  the  worker.  With  the  disengaged  fingers  of 
the  hand  holding  the  needle,  the  cotton  plug  is  removed 
from  the  tube  by  a  twisting  motion  and  placed  between 
the  index  and  second  fingers  of  the  hand  holding  the 
tube,  in  such  a  way  that  the  portion  of  the  plug  which 
fits  into  the  mouth  of  the  test-tube  looks  toward  the 
dorsal  surface  of  the  hand  and  does  not  touch  any  portion 
of  the  hand — this  is  accomplished  by  placing  only  the 
overhanging  portion  of  the  plug  between  the  fingers. 
The  needle  containing  the  bit  of  colony  is  now  to  be 
thrust  into  the  medium  in  the  tube  if  a  stab  culture  is 
desired,  or  rubbed  gently  over  its  surface  if  a  smear 
culture  is  to  be  made.  The  needle  is  then  withdrawn, 
the  cotton  plug  replaced,  and  the  needle  sterilized  before 
it  is  laid  down.  Neither  the  needle  nor  its  handle 
should  touch  the  inner  sides  of  the  test-tube  if  it  can  be 
avoided. 

The  tube  is  then  labelled  and  set  aside  for  observa- 
tion. The  growth  which  appears  in  the  tube  after 


132  BACTERIOLOGY. 

twenty-four  to  thirty-six  hours  will  be  a  pure  culture 
of  the  organisms  of  which  the  colony  was  composed. 

Cultures  of  this  form  are  not  only  useful  as  a  means 
of  preserving  the  different  organisms  with  which  we  may 
be  working,  but  serve  also  to  bring  out  certain  charac- 
teristics of  different  organisms  when  grown  in  this  way. 

If  gelatin  be  employed  and  the  organism  which  has 
been  introduced  into  it  possesses  the  power  of  bringing 
about  liquefaction,  it  will  soon  be  discovered  that  this 
result  is  by  no  means  of  the  same  appearance  for  all 
organisms.  Some  organisms  cause  a  liquefaction  which 
spreads  across  the  whole  upper  surface  of  the  gelatin  and 
continues  gradually  downward ;  again  it  occurs  in  a 
funnel  shape,  the  broad  end  of  the  funnel  being  upper- 
most and  the  point  downward,  corresponding  to  the 
track  of  the  needle.  At  times  a  stocking-  or  sac- formed 
liquefaction  may  be  noticed. 

NOTE. — Obtain  a  number  of  organisms  from  different 
sources  in  pure  cultures  by  the  method  given.  Plant 
them  as  pure  cultures,  all  at  the  same  time,  in  gelatin 
— preferably  gelatin  of  the  same  making — retain  them 
under  the  same  conditions  of  temperature,  and  sketch 
the  finer  differences  in  the  way  in  which  liquefaction 
occurs. 


Of  THE 

UNIVERSITY 

OF 


CHAPTER   X. 

Methods  of  staining— Solutions  employed— Preparation  and  staining  of 
cover-slips — Preparation  of  tissues  for  section -cutting — Staining  of  tissues — 
Special  staining  methods. 

THE  entire  list  of  solutions  and  methods  that  are 
recommended  for  the  staining  of  bacteria  are  not  essen- 
tial to  the  work  of  the  beginner,  so  that  only  those 
which  are  of  most  common  application  will  be  given  in 
this  book.  In  general,  it  suffices  so  say  bacteria  stain 
best  with  watery  solutions  of  the  basic  aniline  dyes, 
and  of  these,  fuchsiu,  gentian-violet,  and  methylene- 
blue  are  those  most  frequently  employed. 

In  practical  work  bacteria  require  to  be  stained  in 
two  conditions ;  either  dried  upon  cover-slips  and  then 
stained,  or  stained  in  sections  of  tissues  in  which  they 
have  been  deposited  during  the  course  of  disease.  In 
both  processes  the  essential  point  to  be  borne  in  mind  is 
that  the  bacteria,  because  of  their  microscopic  dimen- 
sions, require  to  be  more  conspicuously  stained  than 
the  surrounding  materials  upon  the  cover-slips  or  in 
the  sections,  otherwise  their  differentiation  is  a  matter 
of  the  greatest  difficulty,  if  not  of  impossibility.  For 
this  reason,  especially  in  the  case  of  section  staining,  it 
frequently  becomes  necessary  to  decolorize  the  tissues 
after  removing  them  from  the  staining  solutions  in 
order  to  render  the  bacteria  more  prominent,  and  for 
this  purpose  special  methods,  which  provide  for  decolor- 
ization  of  the  tissues  without  robbing  the  bacteria  of 


134  BACTERIOLOGY. 

their  color,  are  employed.  The  ordinary  method  of 
cover-slip  examination  of  bacteria,  constantly  in  use  in 
these  studies,  is  performed  in  the  following  way : 

COVER-SLIP  PREPARATIONS. — In  order  that  the  dis- 
tribution of  the  organisms  upon  the  cover-slips  may  be 
uniform  and  in  as  thin  a  layer  as  possible,  it  is  essential 
that  the  slip  should  be  clean  and  free  from  grease. 
For  cleansing  the  slips  several  methods  may  be  em- 
ployed. 

The  simplest  plan  with  new  cover-slips  is  to  immerse 
them  for  a  few  hours  in  strong  nitric  acid,  after  which 
they  are  rinsed  in  water,  then  in  alcohol,  ether,  and, 
finally,  they  may  be  kept  in  alcohol  to  which  a  little 
ammonia  has  been  added.  When  they  are  to  be  used 
they  should  be  wiped  dry  with  a  clean  cotton  or  silk 
handkerchief, 

If  the  slips  have  been  previously  used,  boiling  in 
strong  soap  solution,  followed  by  rinsing  in  clean  warm 
water,  then  treated  as  above,  renders  them  clean  enough 
for  ordinary  purposes. 

A  method  commonly  employed  is  to  remove  all 
coarse  adherent  matter  from  slips  and  slides  by  allowing 
them  to  remain  for  a  time  in  strong  nitric  or  sulphuric 
acid.  They  are  removed  from  the  acid  after  several 
days,  rinsed  off  in  water,  and  treated  as  above.  Knauer 
has  recently  suggested  the  boiling  of  soiled  cover- slips 
and  slides  for  from  twenty  to  thirty  minutes  in  a  10  per 
cent,  watery  solution  of  lysol,  after  which  they  are  to 
be  carefully  rinsed  in  water  until  all  trace  of  the  lysol 
has  disappeared.  They  are  then  to  be  wiped  dry  with 
a  clean  handkerchief. 

Loeffler's  method,  which  provides  for  the  complete 
removal  of  all  grease,  is  to  warm  the  cover-slips  in  con- 


COVER-SLIP  PREPARATIONS.  135 

centrated  sulphuric  acid  for  a  time,  then  rinse  them  in 
water,  after  which  they  are  kept  in  a  mixture  of  equal 
parts  of  alcohol  and  ammonia.  They  are  to  be  dried 
on  a  cloth  from  which  the  fat  has  been  extracted. 

Steps  in  making  the  preparations.  Place  upon  the 
centre  of  one  of  the  clean,  dry  cover-slips  a  very  small 
drop  of  distilled  water  or  physiological  salt  solution. 
With  a  platinum  needle,  which  has  been  sterilized  in 
the  gas-flame  just  before  using  and  allowed  to  cool,  take 
up  a  very  small  portion  of  the  colony  to  be  examined 
and  mix  it  carefully  with  the  drop  on  the  slip  until 
there  exists  a  very  thin  homogeneous  film  over  the 
larger  part  of  the  surface.  This  is  to  be  dried  upon 
the  slip  by  either  allowing  it  to  remain  upon  the  table 
in  the  horizontal  position  under  a  cover,  to  protect  it 
from  dust,  or  by  holding  it  between  the  fingers  (not  with 
the  forceps),  at  some  distance  above  the  gas-flame,  until 
it  is  quite  dry.  If  held  with  the  forceps  over  the  flame 
at  this  stage,  too  much  heat  may  be  unconsciously  ap- 
plied, and  the  morphology  of  the  organisms  in  the  prep- 
aration distorted.  When  held  between  the  fingers  with 
the  layer  of  bacteria  away  from  the  flame  no  such  acci- 
dent is  likely  to  occur.  When  the  whole  pellicle  is  com- 
pletely dried  the  slip  is  to  be  taken  up  with  the  forceps, 
and,  holding  the  side  upon  which  the  bacteria  are  de- 
posited away  from  the  direct  action  of  the  flame,  is  to 
be  passed  through  the  flame  three  times,  a  little  more 
than  one  second  being  allowed  for  each  transit.  Unless 
the  preliminary  drying  at  the  low  temperature  has  been 
complete,  the  preparation  will  be  rendered  worthless  by 
the  subsequent  " fixing"  at  the  higher  temperature,  for 
the  reason  that  the  protoplasm  of  bacteria  when  moist 
coagulates  at  these  temperatures,  and  in  doing  so  the 


136  BACTERIOLOGY. 

Dormal  outline  of  the  cells  is  altered.  If  carefully  dried 
before  fixing,  this  does  not  occur  and  the  morphology 
of  the  organism  remains  unchanged.  A  better  plan  for 
the  process  of  fixing  is  to  employ  a  copper  plate  of  about 
35  cm.  long  by  10  cm.  wide  by  0.3  cm.  thick.  This 
plate  is  laid  upon  an  iron  tripod  and  a  small  gas- flame 
is  placed  beneath  one  of  its  extremities.  By  this  ar- 
rangement one  can  get  a  graduated  temperature,  begin- 
ning at  the  point  of  the  plate  above  the  gas-flame  where 
it  is  hottest,  and  becoming  gradually  cooler  toward  the 
other  end  of  the  plate,  which  may  be  of  a  very  low  tem- 
perature. By  dropping  water  upon  the  plate,  beginning 
at  the  hottest  point  and  proceeding  toward  the  cooler 
end,  it  is  easy  to  determine  the  point  at  which  the  water 
just  boils ;  it  is  at  a  little  below  this  point  that  the 
cover-slips  are  to  be  placed,  bacteria  side  up,  and  allowed 
to  remain  about  ten  minutes,  when  the  fixing  will  be 
complete.  The  same  may  be  accomplished  in  a  small 
copper  drying  oven,  which  is  regulated  to  remain  at  the 
temperature  of  95°  to  98°  C.  In  very  particular  work 
this  plan  is  to  be  preferred  to  the  process  of  passing  the 
cover- slips  through  the  flame,  as  the  organisms  are 
always  subjected  to  the  same  degree  of  heat,  and  the 
distortions  which  sometimes  occur  from  the  too  great 
and  irregular  application  of  high  temperatures  may  in 
part  be  eliminated,  or  if  not,  will  be  more  nearly  con- 
stant. The  fixing  consists  in  drying  or  coagulating  the 
gelatinous  envelope  surrounding  the  organisms,  by  which 
means  they  are  caused  to  adhere  to  the  surface  of  the 
cover-slip.  When  fixed,  the  staining  is  usually  a  simple 
matter.  The  majority  of  bacteria  with  which  the  be- 
ginner will  have  to  deal  stain  readily  with  solutions  of 
any  of  the  basic  aniline  dyes. 


CO  VER-SLIP  PREPAEA TIONS.  137 

To  stain  the  fixed  cover-slip  preparation  it  is  taken 
by  one  of  its  edges  between  the  forceps,  and  a  few 
drops  of  a  watery  solution  of  fuchsin,  gentian- violet,  or 
methylene-blue  are  placed  upon  the  film  and  are  allowed 
to  remain  there  twenty  to  thirty  seconds.  The  slip  is 
then  carefully  rinsed  in  water,  and  without  drying  is 
placed  bacteria  down  upon  a  slide ;  the  excess  of  water 
is  taken  up  by  covering  it  with  blotting-paper  and  gently 
pressing  upon  it,  and  the  preparation  is  ready  for  exam- 
ination. 

Another  plan  that  is  sometimes  used  is  to  bring  the 
slip  upon  the  slide,  bacteria  down,  without  rinsing  off 
the  staining  fluid ;  the  excess  of  fluid  is  removed  with 
blotting-paper  and  the  preparation  is  ready  for  examina- 
tion with  the  microscope.  This  method  is  satisfactory 
and  time-saving,  but  must  always  be  practised  with  care. 
The  staining  fluid  should  always  be  carefully  filtered  be- 
fore using,  to  rid  it  of  insoluble  particles  which  might  be 
taken  for  bacteria.  If  upon  examination  the  preparation 
proves  to  be  of  particular  interest,  so  that  it  is  desirable 
to  preserve  it,  then  it  is  to  be  mounted  permanently.  The 
drop  of  immersion  oil  is  to  be  removed  from  the  surface 
of  the  slip  with  blotting-paper,  and  the  slip  loosened,  or 
rather  floated,  from  the  slide  by  allowing  water  to  float 
around  its  edges.  It  is  then  taken  up  with  the  forceps, 
carefully  deprived  of  the  water  adhering  to  it  by  means 
of  blotting-paper  and  then  allowed  to  dry.  When  dry 
it  is  mounted  in  xylol-Canada-balsam  by  placing  a  small 
drop  of  the  balsam  upon  the  surface  of  the  film,  and 
then  inverting  the  slip  upon  a  clean  glass  slide.  It  is 
sometimes  desirable  to  have  the  balsam  harden  quickly, 
and  a  method  that  is  commonly  employed  to  induce  this 
is  as  follows  :  The  slide,  held  by  one  of  its  ends  between 


138  BACTERIOLOGY. 

the  fingers,  is  warmed  over  the  gas-flame  until  quite 
hot ;  a  drop  of  balsam  is  then  placed  on  the  centre  of 
it,  and  it  is  again  warmed  ;  the  cover-slip  is  then  placed 
in  position,  and  when  the  balsam  is  evenly  distributed 
the  temperature  is  rapidly  reduced  by  rubbing  the  bot- 
tom of  the  slide  with  a  towel  soaked  in  cold  water. 
Usually  the  preparation  is  firmly  fixed  after  this  treat- 
ment ;  a  little  practice  is  necessary,  however,  in  order 
not  to  overheat  and  not  to  crack  the  slide.  The  method 
is  applicable  only  to  cover-slip  preparations,  and  cannot 
be  used  with  tissues. 

IMPRESSION  COVER-SLIP  PREPARATIONS. — The  im- 
pression preparations  differ  in  value  from  the  ordinary 
cover-slip  preparations  only  in  one  respect ;  they  pre- 
sent an  impression  of  the  organisms  as  they  were  ar- 
ranged in  the  colony  from  which  the  preparation  is 
made.  They  are  made  by  gently  covering  the  colony 
with  a  thin,  clean  cover-slip,  lightly  pressing  upon  it, 
and,  without  moving  the  slip  laterally,  lifting  it  up  by 
one  of  its  edges.  The  organisms  adhere  to  the  slip  in 
the  same  relation  to  one  another  that  they  had  in  the 
colony.  The  subsequent  steps  of  drying,  fixing,  stain- 
ing, and  mounting  are  the  same  as  those  just  given  for 
the  ordinary  cover-slip  preparations. 

By  this  method  constancies  in  the  arrangement  and 
grouping  of  the  individuals  in  a  colony  can  often  be 
made  out.  Some  will  always  appear  irregularly  massed 
together,  others  will  grow  in  parallel  bundles,  while 
others,  again,  will  be  seen  as  long  twisted  threads. 

NOTE. — From  a  colony  of  bacillus  subtilis  make  a 
cover-slip  preparation  in  the  ordinary  way ;  now  make 
an  impression  cover-slip  of  another  colony  of  the  same 
organism.  Compare  the  results. 


THE  ORDINARY  STAINING  SOLUTIONS.       139 

THE  OKDINARY  STAINING  SOLUTIONS. — The  solu- 
tions commonly  employed  in  staining  cover-slip  prepa- 
rations are,  as  has  been  stated,  watery  solutions  of  the 
basic  aniline  dyes — fuchsin,  gentian-violet,  and  methy- 
lene-blue.  These  solutions  may  be  prepared  either  by 
directly  dissolving  the  dyes  in  substance  in  water  until 
the  proper  degree  of  concentration  has  been  reached,  or 
by  preparing  them  from  concentrated  watery  or  alco- 
holic solutions  of  the  dyes  which  may  be  kept  on  hand 
as  stock.  The  latter  method  is  that  commonly  prac- 
tised. 

The  solutions  of  the  colors  which  are  in  constant  use 
in  staining  are  prepared  as  follows  : 

Prepare  as  stock,  saturated  alcoholic  or  watery  solu- 
tions of  fuchsin,  gentian-violet,  and  methylene-blue. 
These  solutions  are  best  prepared  by  pouring  into  clean 
bottles  enough  of  the  dyes  in  substance  to  fill  them  to 
about  one-fourth  of  their  capacity.  The  bottle  should 
then  be  filled  with  alcohol  or  with  water,  tightly  corked, 
well  shaken,  and  allowed  to  stand  for  twenty-four  hours. 
If  at  the  end  of  this  time  all  the  staining  material  has 
been  dissolved,  more  should  be  added,  the  bottle  being 
again  shaken,  and  allowed  to  stand  for  another  twenty- 
four  hours ;  this  must  be  repeated  until  a  permanent 
sediment  of  undissolved  coloring  matter  is  seen  upon 
the  bottom  of  the  bottle.  This  will  then  be  labelled 
saturated  alcoholic  or  watery  solution  of  fuchsin,  gen- 
tian-violet, or  methylene-blue,  as  the  case  may  be.  The 
alcoholic  solutions  are  not  employed  for  staining  purposes. 

The  solutions  with  which  the  staining  is  accomplished 
are  made  from  these  stock  solutions  in  the  following 
way  : 

An  ordinary  test-tube  of  about  13  mm.  diameter  is 


140 


BACTERIOLOGY. 


three-fourths  filled  with  distilled  water  and  the  concen- 
trated alcoholic  or  watery  solution  of  the  dye  is  then 
added,  little  by  little,  until  one  can  just  see  through  the 
solution.  It  is  then  ready  for  use.  Care  must  be  taken 
that  the  color  does  not  become  too  dense.  The  best  re- 
sults are  obtained  when  it  is  just  transparent  as  viewed 
through  a  layer  of  about  12  to  14  mm.  thick. 

These  represent  the  staining  solutions  in  everyday 
use.  They  are  kept  in  bottles  supplied  with  stoppers 
and  pipettes  (Fig.  31),  and  when  used  are  dropped  upon 

FIG.  31. 


Rack  of  bottles  for  staining  solutions. 

the  preparation  to  be  stained.  After  remaining  upon 
the  preparation  for  from  twenty  to  thirty  seconds,  they 
are  washed  off  in  water,  and  the  preparation  can  then 
be  examined. 

For  certain  bacteria  which  stain  only  imperfectly 
with  these  simple  solutions  it  is  necessary  to  employ 
some  agent  that  will  increase  the  penetrating  action  of 
the  dyes.  Experience  has  taught  us  that  this  can  be 
accomplished  by  the  addition  to  the  solutions  of  small 
quantities  of  alkaline  substances  or  by  dissolving  the 
staining  materials  in  strong  watery  solutions  of  either 


THE  ORDINARY  STAINING  SOLUTIONS.       141 

aniline  oil  or  carbolic  acid,  instead  of  simple  water — in 
other  words,  by  employing  mordants  with  the  stains. 

Of  the  solutions  thus  prepared  which  may  always  be 
employed  upon  bacteria  that  show  a  tendency  to  stain 
imperfectly,  there  are  three  in  common  use — Loeffler's 
alkaline  methylene-blue  solution ;  the  Koch-Ehrlich 
aniline-water  solution  of  either  fuchsin,  gentian-violet, 
or  methylene-blue,  and  ZiehPs  solution  of  fuchsin  in 
carbolic  acid.  These  solutions  are  as  follows  : 

Lceffler's  alkaline  methylene-blue  solution  : 

Concentrated  alcoholic  solution  of  methylene-blue     30  c.c. 
Caustic  potash  in  1 : 10,000  solution       .       .       .       .100  c.c. 

Koch-Ehrlich  aniline-water  solutions.  To  about  100 
c.c.  of  distilled  water  aniline  oil  is  added,  drop  by  drop, 
and  the  solution  thoroughly  shaken  after  each  addition, 
until  it  is  of  an  opaque  appearance.  It  is  then  filtered 
through  moistened  filter-paper  until  the  filtrate  is  per- 
fectly clear.  To  100  c.c.  of  the  clear  filtrate  add  10  c.c. 
of  absolute  alcohol  and  11  c.c.  of  the  concentrated  al- 
coholic solution  of  either  fuchsin,  methylene-blue,  or 
gentian-violet,  preferably  fuchsin  or  gentian-violet. 

Ziehl's  carbolic-juchsin  solution  : 

Distilled  water 100  c.c. 

Carbolic  acid  (crystalline) 5  grammes. 

Alcohol    .  10  c.c. 

Fuchsin  in  substance 1  gramme. 

Or  it  may  be  prepared  by  adding  to  a  5  per  cent, 
watery  solution  of  carbolic  acid  the  saturated  alcoholic 
solution  of  fuchsin  until  a  metallic  lustre  appears  on  the 
surface  of  the  fluid. 

The  Koch-Ehrlich  solution  decomposes  after  having 
been  made  for  a  time,  so  that  it  is  better  to  prepare 
it  fresh  when  needed  in  small  quantities  than  to  ein- 

7* 


142  BACTERIOLOGY. 

ploy  old  solutions.  Solutions  older  than  fourteen  days 
should  not  be  used. 

The  three  solutions  just  given  may  be  used  for  cover- 
glass  preparations  in  the  ordinary  way. 

In  some  manipulations  it  becomes  necessary  to  stain 
the  bacteria  very  intensely,  so  that  they  may  retain  their 
color  when  exposed  to  the  action  of  decolorizing  agents. 
These  methods  are  usually  employed  when  it  is  desirable 
to  deprive  surrounding  objects  or  tissues  of  their  color,  in 
order  that  the  stained  bacteria  may  stand  out  in  greater 
contrast.  It  is  in  these  cases  that  the  staining  solu- 
tion with  which  the  bacteria  are  being  treated  is  to  be 
warmed,  and  in  some  cases  boiled,  so  as  to  further 
increase  its  penetrating  action.  When  so  treated,  cer- 
tain of  the  bacteria  will  retain  their  color,  even  when 
exposed  to  very  strong  decolorizers.  The  tubercle 
bacillus  is  characterized  from  all  other  bacteria,  except 
the  bacillus  of  leprosy,  by  the  tenacity  with  which  it 
retains  it  color  when  treated  in  this  way.  It  is  an 
organism  that  is  difficult  to  stain,  but  when  once  stained 
is  equally  difficult  to  rob  of  its  color. 

METHOD  OF  STAINING  THE  TUBERCLE  BACILLUS.— 
Select  from  the  sputum  of  a  tuberculous  subject  one  of 
the  small,  white,  cheesy  masses  which  it  is  seen  to  con- 
tain. Spread  this  upon  a  cover-slip  and  dry  and  fix 
it  in  the  usual  way.  The  slip  is  now  to  be  taken  by 
its  edge  with  the  forceps  and  the  film  covered  with  a 
few  drops  of  either  the  solution  of  Koch-Ehrlich  or  of 
Ziehl.  It  is  then  held  over  the  gas-flame,  at  first  some 
distance  away,  gradually  being  brought  nearer,  until  the 
fluid  begins  to  boil.  After  it  has  bubbled  up  once  or 
twice  it  is  removed  from  the  flame,  the  excess  of  stain- 
ing washed  away  in  a  stream  of  water,  and  it  is  then 


STAINING  THE  TUBERCLE  BACILLUS.         143 

immersed  in  a  30  per  cent,  solution  of  nitric  acid  in 
water  and  allowed  to  remain  there  until  all  the  color 
has  disappeared.  In  some  cases  this  takes  longer  than 
in  others.  One  can  always  determine  if  decolorization 
is  complete  by  washing  off  the  acid  in  a  stream  of  water. 
If  the  preparation  is  still  quite  colored  it  should  be  again 
immersed  in  the  acid ;  if  of  only  a  very  faint  color  it 
may  be  dipped  in  alcohol,  again  washed  off  in  water,  and 
may  now  be  stained  with  some  contrast  color.  If,  for 
example,  the  tubercle  bacilli  have  been  stained  with 
fuchsin,  methylene-blue  forms  a  good  contrast  stain.  In 
making  the  contrast  stain  the  steps  in  the  process  are 
exactly  those  followed  in  the  ordinary  staining  of  cover- 
slip  preparations  in  general :  the  slip  containing  the 
stained  tubercle  bacilli  is  rinsed  off  carefully  in  water, 
and  a  few  drops  of  the  me  thy  leu  e- blue  solution  are 
placed  upon  it  and  allowed  to  remain  for  thirty  to  forty 
seconds,  when  it  is  again  rinsed  in  water  and  examined 
microscopically.  For  the  purpose  of  observing  the 
difference  between  the  behavior  of  the  tubercle  bacilli 
and  the  other  organisms  present  in  the  preparation 
toward  this  method  of  staining,  it  is  well  to  examine 
the  preparation  microscopically  before  the  contrast  stain 
is  made,  then  remove  it,  give  it  the  contrast  color,  and 
examine  it  again.  It  will  be  seen  that  before  the  con- 
trast color  has  been  given  to  the  preparation  the 
tubercle  bacilli  will  be  the  only  stained  objects  to  be 
made  out,  and  the  preparation  will  appear  devoid  of 
other  organisms,  but  upon  examining  it  after  it  has 
received  the  contrast  color  a  great  many  other  or- 
ganisms will  now  appear  ;  these  will  take  on  the  second 
color  employed,  while  the  tubercle  bacilli  will  retain 
their  original  color.  Before  decolorization  all  organisms 


144  BACTERIOLOGY. 

in  the  preparation  were  of  the  same  color,  but  during  the 
application  of  the  decolorizing  solution  all  except  the 
tubercle  bacilli  gave  up  their  color.  This  characteristic, 
together  with  reactions  to  be  described,  as  said,  serves  to 
differentiate  the  tubercle  bacillus  from  other  organisms 
with  which  it  might  be  confounded.  A  number  of 
different  methods  have  been  suggested  for  the  staining 
of  tubercle  bacilli,  but  the  original  method  as  employed 
by  Koch  is  so  satisfactory  in  its  results  that  it  is  not 
advisable  to  substitute  others  for  it.  The  above  differs 
from  the  original  Koch-Ehrlich  method  for  the  staining 
of  tubercle  bacilli  in  sputum  only  in  the  occasional  em- 
ployment of  ZiehFs  carbolic-fuchsin  solution  and  in  the 
method  of  heating  the  preparation  with  the  staining  fluid 
upon  it. 

As  Nuttall  has  pointed  out,  however,  the  strong  acid 
decolorizer  used  in  this  method  can,  with  advantage,  be 
replaced  by  much  more  dilute  solutions,  as  a  certain 
number  of  the  bacilli  are  entirely  decolorized  by  the  too 
energetic  action  of  the  strong  acids.  He  recommends 
the  following  method  of  decolorization  :  After  staining 
the  slip  or  section  in  the  usual  way,  pass  it  through  three 
alcohols ;  it  is  then  to  be  washed  out  in  a  solution  com- 
posed of 

Water 150  c.c. 

Alcohol 50  c.c. 

Concen.  sulphuric  acid 20  to  30  drops. 

From  this  it  is  removed  to  water  and  carefully  rinsed. 
The  remaining  steps  in  the  process  are  the  same  as  those 
given  in  the  other  methods. 

GABBETT'S  METHOD  for  the  staining  of  tubercle 
bacilli  recommends  itself  because  of  its  simplicity  and 
the  rapidity  with  which  it  can  be  performed.  By  many 


GRAM'S  METHOD.  145 

it  is  considered  the  best  method  for  routine  employment. 
It  consists  in  staining  the  cover-slips,  prepared  in  the 
manner  given,  for  from  two  to  five  minutes  in  a  cold 
carbolic-fuchsin  solution,  after  which  they  are  subjected 
to  the  action  of  Gabbett's  methylene-blue  sulphuric  acid 
solution.  This  latter  consists  of 

Sulphuric  acid,  strength  25  per  cent          .    100  c.c. 
Methylene-blue,  in  substance      .       .       .       1  to  2  grammes. 

They  are  then  rinsed  off  in  water  and  are  ready  for  ex- 
amination. The  tubercle  bacilli  will  be  stained  red  by 
the  fuchsin,  while  all  other  bacteria,  cell  nuclei,  etc.,  will 
be  tinted  blue. 

GRAM'S  METHOD. — Another  differential  method  of 
staining  which  is  very  commonly  employed  is  that 
known  as  Gram's  method.  In  this  method  the  objects 
to  be  stained  are  treated  with  an  aniline-water  solution 
of  gentian-violet  made  after  the  formula  of  Koch- 
Ehrlich.  After  remaining  in  this  for  twenty  to  thirty 
minutes  they  are  immersed  in  an  iodine  solution  com- 
posed of 

Iodine 1  gramme. 

Potassium  iodide 2  grammes. 

Distilled  water     .......      300  c.c. 

In  this  they  remain  for  about  five  minutes ;  they  are 
then  transferred  to  alcohol  and  thoroughly  rinsed.  If 
they  are  still  of  a  violet  color  they  are  again  treated 
with  the  iodine  solution  followed  by  alcohol,  and  this  is 
continued  until  no  trace  of  violet  color  is  visible  to  the 
naked  eye.  They  may  then  be  examined,  or  a  contrast 
color  of  carmine  or  Bismarck-brown  may  be  given  them. 

This  method  is  particularly  useful  in  demonstrating 
the  capsule  which  is  seen  to  surround  some  bacteria, 
particularly  the  micrococcus  lanceolatus  of  pneumonia. 


146  BACTERIOLOGY. 

GLACIAL  ACETIC  ACID  METHOD. — Another  method 
which  may  be  employed  for  demonstrating  the  presence 
of  the  capsule  surrounding  certain  organisms,  is  to  pre- 
pare the  cover-slips  in  the  ordinary  way,  then  cover  the 
layer  of  bacteria  upon  them  with  glacial  acetic  acid, 
which  is  instantly  poured  off  (not  washed  off  in  water), 
and  the  aniline-water  gentian-violet  solution  dropped 
upon  them ;  this  is  allowed  to  remain  three  or  four 
minutes,  is  poured  off,  and  a  few  drops  more  are  added, 
and  lastly  the  slip  is  washed  off  in  water.  A  very  clear, 
sharply  cut  picture  usually  follows  this  method  of  pro- 
cedure. 

STAINING  OF  SPORES. — We  have  learned  that  one  of 
the  points  by  which  spores  may  be  recognized  is  their 
refusal  to  take  up  staining  substances  when  applied  in 
the  ordinary  way.  They  may,  however,  be  stained  by 
special  methods  ;  of  these  one  that  has  given  very  satis- 
factory results  in  our  hands  is  as  follows :  The  cover- 
slip  is  to  be  prepared  from  the  material  containing  the 
spores  in  the  ordinary  way,  dried,  and  fixed.  It  is  then 
to  be  held  by  its  edge  with  the  forceps,  and  its  surface 
covered  with  Loeffler's  alkaline  methylene-blue  solu- 
tion. It  is  then  held  over  the  Bunsen  flame  until  the 
fluid  boils ;  it  is  then  removed,  and  after  a  few  seconds 
is  heated  again.  This  is  continued  for  about  one  min- 
ute, after  which  it  is  washed  off  in  water  and  dipped 
five  or  six  times  in  alcohol  containing  about  0.2  to  0.3 
per  cent,  of  hydrochloric  acid.  This  is  rinsed  off  in 
water  and  the  preparation  is  now  stained  for  from  eight 
to  ten  seconds  in  aniline-water  fuchsin  solution  (Koch- 
Ehrlich  solution),  and  finally  again  washed  in  water. 
By  this  method  the  spores  are  of  a  blue  color  and  the 
body  of  the  cell  red. 


MOELLER'S  METHOD  FOE  STAINING  SPORES.     147 

By  another  process  the  cover-slip  is  floated,  bacteria 
down,  upon  the  surface  of  freshly  prepared  Koch-Ehrlich 
solution  of  fuchsin  contained  in  a  watch-crystal.  This  is 
then  held  by  its  edge  with  the  forceps  about  2  cm.  above 
a  very  small  flame  of  a  Buusen  burner,  care  being  taken 
that  the  flame  touches  only  the  centre  of  the  bottom  of 
the  crystal.  After  a  few  seconds  the  crystal  is  elevated 
gradually  until  it  is  about  6  to  8  cm.  above  the  flame, 
then  it  is  slowly  moved  down  to  the  flame  again,  and 
this  up-and-down  movement  is  continued  until  the  stain- 
ing fluid  begins  to  boil.  As  soon  as  a  few  bubbles  have 
been  given  off  it  is  held  aside  for  a  minute  or  two  and 
the  process  of  heating  is  repeated.  When  the  boiling 
begins  the  crystal  is  held  aside  again  for  a  minute  or 
two.  The  crystal  is  heated  in  this  way  for  about  five 
or  six  consecutive  times.  When  the  fluid  has  stood  for 
about  five  minutes  after  the  last  boiling,  the  preparation 
is  transferred,  without  washing  in  water,  into  a  second 
watch-crystal  containing  the  following  decolorizing  solu- 
tion : 

Absolute  alcohol 100  c.c. 

Hydrochloric  acid    .       .       , 3  c.c. 

In  this  solution  it  is  placed,  bacteria  up,  and  the 
vessel  is  tilted  from  side  to  side  for  about  one  minute. 
It  is  then  removed,  washed  in  water,  and  stained  with 
the  methylene-blue  solution.  The  spores  will  be  stained 
red  and  the  body  of  the  cells  will  be  blue. 

MOELLER'S  METHOD  FOR  STAINING  SPORES. — A 
method  that  has  recently  been  published  by  Moeller  is 
designed  to  favor  the  penetration  of  the  coloring  mate- 
rial through  the  spore  membrane  by  macerating  the 
spores  in  a  solution  of  chromic  acid  before  staining 
them.  It  is  as  follows  : 


148  BACTERIOLOGY. 

The  cover-slips  are  prepared  in  the  usual  way,  or  the 
fixing  may  be  accomplished  with  absolute  alcohol  in- 
stead of  high  temperatures.  The  preparation  is  then 
held  for  two  minutes  in  chloroform,  then  washed  off  in 
water,  then  placed  for  from  one-half  to  two  minutes  in 
a  5  per  cent,  solution  of  chromic  acid ;  again  washed 
off  in  water,  and  now  stained  in  carbolic  fuchsin.  In 
the  process  of  staining,  the  slip  is  taken  by  the  corner 
with  the  forceps,  and  carbolic  fuchsin  is  dropped  upon 
the  side  containing  the  spores.  It  is  then  held  over  the 
flame  until  it  boils,  and  then  held  some  distance  above 
the  flame  for  one  minute.  The  staining  fluid  is  then 
poured  off  and  the  preparation  is  completely  decolorized 
in  5  per  cent,  sulphuric  acid,  again  washed  off  in  water, 
and  finally  stained  for  thirty  seconds  in  the  watery 
methylene-blue  solution.  The  spores  will  be  red,  the 
body  of  the  cells  blue. 

In  this  method  the  object  of  the  preliminary  ex- 
posure to  chloroform  is  to  dissolve  away  any  crystals 
of  lecithin,  cholesterin,  or  fat  that  may  be  in  the  prepa- 
ration, and  which  when  stained  might  give  rise  to  con- 
fusion. 

It  must  be  remembered  that  there  are  conspicuous 
differences  in  the  behavior  of  spores  of  different  bac- 
teria to  staining  methods.  Some  stain  readily  by  either 
of  the  methods  especially  devised  for  this  purpose, 
while  others  can  hardly  be  stained  at  all,  or  only  with 
the  greatest  difficulty,  by  any  of  the  known  processes. 

LCEFFLER'S  METHOD  FOR  STAINING  FLAGELLA. — 
For  the  demonstration  of  the  locomotive  apparatus 
possessed  by  motile  bacteria  we  are  indebted  to  Locffler. 
By  a  special  method  of  staining  in  which  the  use  of 
mordants  played  the  essential  part,  he  has  shown  that 


STAINING  FLAGELLA.  149 

these  organisms  possess  very  delicate,  hair-like  appen- 
dages, by  the  lashing  movements  of  which  they  propel 
themselves  through  the  fluid  in  which  they  are  located. 
The  method  as  given  by  Lreffler  is  as  follows  : 

(1)  It  is  essential  that  the  bacteria  be  evenly  and  not 
too  numerously  distributed  upon  the  cover-slip.  The 
slips  must  therefore  be  carefully  cleansed.  (See  Lceffler's 
method  of  cleaning  cover-slips.)  Five  or  six  of  the 
carefully  cleansed  cover-slips  are  to  be  placed  in  a  line 
on  the  table,  and  on  the  centre  of  each  slip  a  very  small 
drop  of  tap-water  is  placed.  From  the  culture  to  be 
examined  a  minute  portion  is  transferred  to  the  first 
slip  and  carefully  mixed  with  the  drop  of  water  ;  from 
this  mixture  a  small  portion  is  transferred  to  the  second, 
and  from  the  second  to  the  third  slip,  and  so  on — in 
this  way  insuring  a  dilution  of  the  number  of  organ- 
isms present  in  the  preparation. 

These  slips  are  then  dried  and  fixed  in  the  ordinary 
way.  They  are  next  to  be  warmed  in  the  following 
solution  : 

Tannic  acid  solution  in  water  (20  acid,  80  water)  .  10  c.c. 
Cold  saturated  solution  of  ferro-sulphate  .  .  .  5  c.c. 
Saturated  watery  or  alcoholic  solution  of  fuchsin  .  1  c.c. 

This  solution  represents  the  mordant.  A  few  drops 
of  it  are  to  be  placed  upon  the  film  of  bacteria  on  the 
cover-slip,  which  is  then  to  be  held  over  the  flame  until 
the  solution  begins  to  steam.  It  should  not  be  boiled. 
After  steaming,  the  mordant  is  washed  off  in  water  and 
finally  in  alcohol.  The  bacteria  are  then  to  be  stained 
in  a  saturated  aniline-water  fuchsin  solution. 

When  treated  in  this  way  different  bacteria  behave 
differently  :  the  flagella  of  some  stain  readily  in  the 
above  solutions  ;  others  require  the  addition  of  an  alkali 


150  BACTERIOLOGY. 

in  varying  quantities ;  while  others  stain  best  after  the 
addition  of  acids.  To  meet  these  conditions  an  exact 
1  per  cent,  solution  of  caustic  soda  in  water  must  be 
prepared,  and  also  a  solution  of  sulphuric  acid  in  water 
of  such  strength  that  one  cubic  centimetre  will  be  exactly 
neutralized  by  one  cubic  centimetre  of  the  alkaline  so- 
lution. 

For  different  bacteria  which  have  been  studied  by 
this  method,  the  one  or  the  other  of  these  solutions  is 
to  be  added  to  the  mordant  in  the  following  propor- 
tions. 

Of  the  acid  solution  : 

For  the  bacillus  of  Asiatic  cholera       .       .       .    %  to  1  drop. 
For  the  spirillum  rubrum 9  drops. 

Of  the  alkaline  solution  : 

For  the  bacillus  of  typhoid  fever  ....    1  c.c. 

For  the  bacillus  subtilis 28  to  30  drops. 

For  the  bacillus  of  malignant  oadema  .       .       .    36  to  37      " 

For  other  organisms  one  must  determine  whether  the 
results  are  better  after  the  addition  of  acid  or  alkali, 
and  how  much  of  either  is  required.  In  general  it  may 
be  said  that  bacteria  which  produce  acids  in  the  media 
in  which  they  are  growing  require  the  addition  of  alka- 
lies to  the  mordant,  while  those  that  produce  alkalies 
require  acids  to  be  added.  By  following  Loeffler's  direc- 
tions the  delicate,  hair-like  flagellaB  on  motile  organisms 
may  be  rendered  plainly  visible. 

There  are  several  points  and  slight  modifications  in 
connection  with  this  method  that  require  to  be  empha- 
sized in  order  to  insure  success  :  The  culture  to  be  em- 
ployed should  be  young,  not  over  18-20  hours  old.  It 
should  have  developed  for  this  time  on  fresh  agar-agar  at 


THE  METHOD  OF  VAN  ERMENGEM.  151 

37°  to  38°  C. ;  the  solutions  should  not  be  perfectly  fresh, 
as  the  best  results  are  obtained  from  the  use  of  old  solu- 
tions that  have  stood,  exposed  to  the  air,  and  that  have 
been  filtered  just  before  using  ;  when  placed  on  the  cover- 
slip  and  held  over  the  flame  never  heat  the  mordant  to  the 
boiling-point ;  indeed,  the  best  results  are  obtained  when  the 
preparation  is  held  high  above  the  flame  and  removed  from 
it  at  the  first  evidence  of  vaporization,  or,  better  still,  a 
little  before  this  point  is  reached.  We  have  derived  no 
advantage  from  the  addition  of  acids  or  alkalies  to  the 
mordant  as  recommended  by  Loeffler,  but  obtain,  with  a 
fair  degree  of  regularity,  satisfactory  results  through  the 
use  of  the  neutral  mordant  alone.1 

BUNGE'S  METHOD. — A  useful  modification  of  Loeffler's 
method  is  that  recommenced  by  Bunge  :  prepare  a  satu- 
rated solution  of  tannin,  and  a  solution  of  liquor  ferri 
sesquichlor.  of  the  strength  of  1  : 20  of  distilled  water. 
To  3  parts  of  the  tannin  solution  add  1  part  of  the  dilute 
iron  solution.  To  10  c.c.  of  such  a  mixture  add  1  c.c. 
of  concentrated  watery  solution  of  fuchsin.  This  mor- 
dant is  not  to  be  used  fresh,  but  only  after  standing  ex- 
posed to  the  air  for  several  days  (better  for  several 
weeks).  After  preparing  the  cover-slip  with  all  pre- 
cautions necessary  to  cleanliness  the  filtered  mordant  is 
allowed  to  act  cold  for  about  five  minutes,  after  which 
it  is  slightly  warmed  ;  the  slip  is  then  washed  off  in 
water,  dried,  and  faintly  stained  with  carbol  fuchsin.  No 
addition  of  acid  or  alkali  to  the  mordant  is  necessary. 

THE  METHOD  OF  VAN  ERMENGEM. — Another 
method  of  demonstrating  the  presence  of  flagella  is 

1  I  am  indebted  to  Dr.  James  Homer  Wright,  Thomas  Scott  Fellow  in  Hy- 
giene, 1892-93,  University  of  Pennsylvania,  for  some  of  the  suggestions  in 
connection  with  the  modification  of  this  method. 


152  BACTERIOLOGY. 

that  suggested  by  Van  Erraengem.  It  is  somewhat 
more  complicated  than  either  of  the  preceding  methods. 
The  steps  in  the  process  are  as  follows  : 

In  the  centre  of  a  perfectly  cleaned  cover- slip  place  a 
drop  of  a  very  dilute  suspension,  in  physiological  salt 
solution,  of  a  10-  to  18-hour  old  agar-agar  culture  of  the 
organism  to  be  studied.  The  suspension  of  the  organ- 
isms in  the  salt  solution  should  be  very  dilute  in  order 
to  favor  the  isolation  of  single  cells  on  the  slip  and  also 
to  obviate  the  occurrence  of  excessive  precipitation.  The 
slips  are  then  to  be  dried  in  the  air  and  in  the  gas-flame 
in  the  usual  manner. 

The  mordant  used  consists  of: 

Osmic  acid  (2  per  cent,  solution) 1  part. 

Tannin  (10-25  per  cent,  solution)       ......       2  parts. 

To  this  4  or  5  drops  of  glacial  acetic  acid  may  be 
added,  but  experience  has  shown  this  to  be  hardly 
necessary. 

Place  a  drop  or  two  of  this  mordant  on  the  cover-slip 
to  be  stained,  and  allow  it  to  act  for  one-half  hour  at 
room  temperature,  or  for  five  minutes  at  50°  to  60°  C. 

Wash  carefully  in  water  and  alcohol,  and  then  im- 
merse for  a  few  seconds  in  the  "  sensitizing  bath,"  viz., 
a  0.25-0.5  per  cent,  solution  of  silver  nitrate.  Without 
washing,  bring  the  slip  into  a  watch-crystalful  of  the 
"reducing  and  reinforcing  bath,"  viz.  : 

Gallic  acid  .       .       .       .       .       .       .  5  grains, 

Tannin •  3      " 

Fused  pot.  acetate 10      " 

Dist.  water 350      " 

After  a  few  seconds  pass  the  slip  back  into  a  watch 
crystal  containing  the  dilute  silver  bath  (0.25-0.5  per 
cent,  solution  of  silver  nitrate  in  water)  and  keep  it  in 


STAINING  IN  GENERAL.  153 

constant  motion  until  the  solution  begins  to  take  on  a 
brown  or  blackish  color.  Wash  in  water  thoroughly ; 
dry  with  blotting  paper,  and  mount  in  balsam. 

STAINING   IN   GENERAL. 

The  physics  of  staining  and  decolorization  is  hardly 
a  subject  to  be  discussed  at  length  in  a  book  of  this 
character ;  but,  as  Kiihne  has-  pointed  out,  it  might  be 
said  that  solutions  which  favor  the  production  of  diffu- 
sion currents  facilitate  intensity  of  staining,  and  by  a 
similar  process  increase  the  energy  of  decolorizing 
agents.  For  example,  tissues  which  are  transferred 
from  water  into  watery  solutions  of  the  coloring  mat- 
ters are  less  intensely  stained  and  more  easily  decolor- 
ized than  when  transferred  from  alcohol  into  watery 
staining  fluids ;  for  the  same  reason  tissues  stained  in 
watery  solutions  of  the  dyes  do  not  become  decolorized 
so  readily  when  placed  in  water  as  when  placed  in  alcohol. 

The  diffusion  of  staining  solutions  into  the  proto- 
plasm of  dried  bacteria,  as  found  upon  cover-slip  prep- 
arations, is  much  greater  and  more  rapid  than  when  the 
same  bacteria  are  located  in  the  interstices  of  tissues. 
These  differences  are  not  in  the  bacteria  themselves,  but 
in  the  obstruction  to  diffusion  offered  by  the  tissues  in 
which  they  are  located. 

The  result  of  absence  of  diffusion  may  easily  be  illus- 
trated. Prepare  a  cover-slip  preparation,  dry  it  care- 
fully, fix  it,  and,  without  allowing  water  to  get  on  it 
from  any  source,  attempt  to  stain  it  with  a  solution  of 
the  dyes  in  absolute  alcohol,  washing  it  out  subsequently 
with  absolute  alcohol ;  the  result  is  negative.  The  ab- 
solute alcohol  does  not  possess  the  property  of  diffusing 


154  BACTERIOLOGY. 

into  the  dried  tissues,  and  hence,  as  has  been  stated 
before,  alcoholic  solutions  of  the  staining  dyes  should 
not  be  employed.  The  staining  dyes  should  always  be 
watery.1 

DECOLORIZING  SOLUTIONS. — As  regards  the  employ- 
ment of  decolorizing  agents,  it  must  always  be  borne  in 
mind  that  objects  which  are  easily  stained  are  also  easily 
decolorized,  and  those  that  can  be  caused  to  take  up  the 
staining  material  only  with  difficulty  are  also  very  diffi- 
cult to  rob  of  their  color.  The  most  common  decolor- 
izer  in  use  is  probably  alcohol — not  absolute  alcohol, 
but  alcohol  containing  more  or  less  of  water.  Water 
alone  has  this  property,  but  in  a  much  lower  degree 
than  dilute  alcohol.  On  the  other  hand,  a  much  more 
energetic  decolorization  than  that  possessed  by  either 
alone  can  be  obtained  by  alternate  exposures  to  alcohol 
and  water.  More  energetic  in  their  decolorizing  action 
than  either  water  or  alcohol  are  solutions  of  the  acids. 
They  appear,  particularly  when  they  are  alcoholic  solu- 
tions, to  diffuse  rapidly  into  tissues  and  bacteria  and 
very  quickly  extract  the  staining  materials  which  have 
been  deposited  there.  For  this  reason  these  solutions 
should  be  employed  with  much  care. 

Very  dilute  acetic  acid  robs  tissues  and  bacteria  of 
their  staining  with  remarkable  activity ;  still  more  ener- 
getic are  solutions  of  the  mineral  acids,  and  particularly, 
as  has  been  said,  when  this  action  is  accompanied  by 
the  decolorizing  properties  of  alcohol. 

1  In  the  beginning  of  this  chapter  it  was  stated  that  the  saturated  alcoholic 
solutions  of  the  dyes  do  not  serve  as  stains  for  bacteria.  It  must  be  remem- 
bered that  this  holds  only  when  absolute  alcohol  and  perfectly  dry  coloring 
matters  have  been  used.  If  but  a  small  proportion  of  water  is  present,  the 
bacteria  may  be  stained  with  these  solutions. 


S1AINING  OF  BACTERIA  IN  TISSUES.         155 

The  acid  solutions  that  are  commonly  employed  are : 

Acetic  acid  in  from  0.1  per  cent,  to  5  per  cent,  watery 
solution. 

Nitric  acid  in  from  20  per  cent,  to  30  per  cent,  watery 
solution. 

Hydrochloric  acid  in  3  per  cent,  solution  in  alcohol. 


STAINING   OF   BACTERIA   IN   TISSUES. 

In  staining  tissues  for  the  purpose  of  demonstrating 
the  bacteria  which  they  may  contain,  a  number  of  points 
must  be  borne  in  mind  :  the  conditions  which  favor  the 
diffusion  of  the  staining  fluids  into  the  bacteria,  are  now 
not  so  favorable  to  rapid  staining  as  they  were  when  the 
bacteria  alone  were  present  upon  cover-slips  ;  the  stain- 
ing of  tissues,  therefore,  requires  a  longer  exposure  to 
the  dyes  than  does  that  of  cover-slips.  In  tissues,  too, 
there  are  other  substances  beside  the  bacteria  which 
become  stained,  and  these,  unless  robbed  in  whole  or  in 
part  of  their  color,  may  so  mask  the  stained  bacteria  as 
to  render  them  difficult,  if  not  impossible,  of  detection. 
Tissues  must,  therefore,  always  be  subjected  to  some 
degree  of  decolorization,  and  this  must  be  practised 
without  depriving  the  bacteria  of  their  color. 

The  details  of  the  methods  of  decolorization  will  be 
described  in  the  section  on  the  technique  of  staining. 

Another  point  to  be  remembered  in  staining  tissues 
is  that  they  can  never  be  heated  and  retain  their  struc- 
ture, in  the  same  way  that  one  heats  cover-slips.  The 
best  results  are  not  obtained  in  efforts  to  hasten  the 
staining  by  subjection  to  high  temperatures,  but  rather 
by  longer  exposures  to  lower  temperatures. 


156  BACTERIOLOGY. 

HARDENING  THE  TISSUES. — The  bits  of  tissue — not 
greater  than  1  cm.  cube — are  to  be  placed,  as  fresh  as 
possible,  in  absolute  alcohol.  The  bit  of  tissue  should 
rest  upon  a  pad  of  cotton  or  filter-paper  in  the  bottle 
containing  the  alcohol,  in  order  that  it  may  be  elevated 
and  surrounded  by  the  part  of  the  alcohol  which  is 
specifically  the  lightest,  and  consequently  contains  least 
water.  The  alcohol  abstracts  water  from  the  tissue,  and, 
as  the  dehydration  proceeds,  the  tissue  becomes  accord- 
ingly more  and  more  dense.  When  of  about  the  con- 
sistency of  fresh  solid  rubber,  or  preferably  not  quite 
so  dense,  it  is  ready  to  cut.  A  small  portion  of  about 
0.5  cm.  cube  should  be  cemented  to  a  bit  of  cork  with 
ordinary  mucilage,  and  allowed  to  remain  in  the  open 
air  for  a  minute  or  two  for  the  mucilage  to  harden. 
Alcohol  should  be  dropped  upon  it  occasionally,  to  pre- 
vent drying  of  the  tissue.  When  the  mucilage  is  hard 
the  cork  with  the  piece  of  tissue  upon  it  may  be  left  in 
alcohol  over  night,  and  on  the  following  day  the  sections 
may  be  cut. 

SECTION-CUTTING. — This  is  accomplished  by  the  use 
of  an  instrument  known  as  a  microtome.  In  Fig.  32 
is  seen  the  form  now  commonly  employed.  It  is 
known  by  the  name  of  the  maker,  as  Schanze's  micro- 
tome. It  is  an  apparatus  provided  with  a  clamp  for 
holding  the  cork  upon  which  the  tissue  is  cemented,  and 
also  a  sliding  clamp  which  carries  a  knife.  The  tissue 
is  clamped  horizontally,  and  the  knife  is  caused  to  slide 
across  its  upper  surface,  also  in  the  horizontal  plane. 
Beneath  the  clamp  for  holding  the  tissue  is  a  milled 
disc,  by  means  of  which  a  screw  is  caused  to  revolve, 
and  in  revolving  raises  or  lowers  the  clamp  holding  the 
tissue,  so  that  the  tissue  may  be  brought  closer  to  or 


SECTION-CUTTING.  157 

farther  from  the  plane  in  which  the  knife  slides.  By 
this  arrangement  sections  of  any  desired  thickness  can 
be  cut  by  turning  the  milled  disc  with  the  one  hand  and 
causing  the  knife  to  traverse  the  tissue  with  the  other. 

FIG.  32. 


Schanze's  microtome. 

The  tissue  and  the  knife-blade  should  be  kept  wet 
with  alcohol,  so  that  the  sections  may  float  upon  the  blade 
of  the  knife,  from  which  they  can  be  easily  removed 
without  tearing,  with  a  curved  needle  or  a  camel-hair 
pencil.  As  the  sections  are  cut  they  are  placed  in  a  dish 
containing  alcohol. 

There  are  some  tissues  which,  by  reason  of  their  his- 
tological  structure,  do  not  become  sufficiently  dense  when 
exposed  to  alcohol  to  permit  of  their  being  cut  in  the 
above  way.  It  becomes  necessary  to  render  them  more 
solid  by  filling  their  interstices  with  some  substance  that 
neither  interferes  with  their  structure,  nor  prevents  their 

8 


158  BACTERIOLOGY. 

being  cut  into  sections.  They  must  be  "imbedded," 
as  this  process  is  called. 

Imbedding  in  celloidin.  Most  convenient  for  this 
purpose  is  celloidin,  a  body  somewhat  similar  to  col- 
lodion, soluble  in  a  mixture  of  equal  parts  of  alcohol 
and  ether,  as  well  as  in  absolute  alcohol. 

After  hardening  in  alcohol  the  tissue  to  be  imbedded 
is  placed  in  a  mixture  of  equal  parts  of  absolute  alcohol 
and  ether  and  left  there  for  twenty-four  hours.  It  is 
then  transferred  to  celloidin.  Two  solutions  of  cel- 
loidin are  to  be  employed,  the  one  a  thin  solution  in 
a  mixture  of  equal  parts  of  absolute  alcohol  and  ether, 
the  other  a  thick  solution  in  the  same  solvent.  Into 
the  thin  solution,  which  should  be  of  about  the  consist- 
ence of  very  thin  syrup,  the  tissue  is  placed  from  the 
absolute  alcohol  and  ether,  and  allowed  to  remain  there 
for  twenty-four  hours.  It  is  then  placed  in  the  thick 
solution  for  about  a  day.  From  this  it  may  be  removed 
and  placed  immediately  upon  a  bit  of  cork  or  a  block 
of  wood.  The  adherent  celloidin  will  act  as  a  cement, 
and  as  it  hardens  rapidly,  the  tissue  is  soon  fast  to  the 
cork.  It  is  then  left  in  60  per  cent,  alcohol  for  twenty- 
four  hours  to  complete  the  solidification  of  the  celloidin, 
after  which  sections  may  be  cut  in  the  way  just  described 
for  tissues  not  so  treated. 

Imbedding  in  paraffin.  After  bits  of  the  tissue  not 
larger  than  a  cubic  centimetre  have  been  hardened  in 
the  usual  way,  they  are  placed  in  fresh  absolute  alcohol 
for  twenty- four  hours  to  complete  the  process.  From 
this  they  are  transferred  to  pure  turpentine,  and  kept 
in  a  warm  oven  at  a  temperatare  not  exceeding  35°  to 
38°  C.  Here  they  remain  for  a  time  sufficient  for  them 
to  become  thoroughly  saturated  with  the  turpentine,  as 


STAINING  OF  THE  SECTIONS.  159 

is  recognized  by  the  transparent  appearance  that  they 
assume.  From  this  they  are  placed  in  paraffin  that  is 
melted  at  53°  C.,  and  allowed  to  remain  in  this  for 
three  or  four  hours.  They  are  then  transferred  to  a 
small  paper  or  metal  mould,  or  a  pill-box,  and  melted 
paraffin  is  poured  over  them.  When  the  paraffin  has 
become  solid  the  mould  or  pill-box  is  removed  from 
around  it,  the  excess  of  paraffin  removed  from  about 
the  imbedded  tissue,  and  the  latter  is  ready  for  cutting. 

When  the  sections  are  cut  they  are  freed  from  par- 
affin by  exposing  them  to  turpentine ;  the  latter  is  re- 
moved by  washing  in  alcohol  and  the  sections  can  now 
be  stained  in  the  ordinary  way.  In  cutting  sections 
from  tissues  that  have  been  imbedded  in  paraffin  the 
long  axis  of  the  knife  should  be  at  nearly  right  angles 
to  the  direction  in  which  the  knife  travels.  For  bac- 
teriological purposes  the  method  of  imbedding  in  par- 
affin does  not  as  a  rule  give  such  good  results  as  when 
the  celloidin  method  is  employed.  In  this  work,  there- 
fore, the  latter  is  usually  preferred. 

STAINING  OF  THE  SECTIONS. — The  sections  when  cut 
may  be  stained  in  a  variety  of  ways.  The  ordinary 
watery  solutions  of  the  three  common  basic  aniline  dyes 
— fuchsin,  gentian-violet,  or  methylene-blue — or,  what 
is  better,  the  alkaline  methylene-blue  solution  of  Loeffler, 
may  be  employed  for  general  use. 

The  acid  aniline  dyes,  as  well  as  some  of  the  vege- 
table coloring  matters,  are  essentially  nuclear  stains,  and 
are  not  applicable  to  the  staining  of  bacteria. 

Into  a  watch-glass  containing  either  of  the  staining 
solutions  mentioned,  the  sections  are  to  be  placed  after 
having  been  in  water  for  about  one  minute.  They 
remain  in  the  staining  solutions  for  from  five  to  eight 


160  BACTERIOLOGY. 

minutes.  They  are  then  removed,  rinsed  in  water,  and 
partly  decolorized  in  0.1  per  cent,  solution  of  acetic  acid  for 
only  a  few  seconds ;  again  washed  out  in  water,  then  in 
absolute  alcohol  for  a  few  seconds,  and  from  this  again 
into  absolute  alcohol  for  the  same  time,  and  finally  into 
cedar  oil  or  xylol.  Here  they  remain  for  from  one-half  to 
three-fourths  of  a  minute.  They  are  now  to  be  carefully 
spread  out  upon  a  spatula,  which  is  held  in  the  fluid 
under  them,  and,  without  draining  off  the  fluid,  are  trans- 
ferred to  a  clean  glass  slide.  This  must  be  done  care- 
fully to  avoid  tearing.  The  easiest  way  to  do  this  is  to 
hold  the  spatula  on  which  the  section  floats  in  one  hand, 
with  its  point  just  touching  the  surface  of  the  glass  slide, 
and  then  with  a  needle  pull  the  section  gently  off  upon 
the  slide.  The  fluid  comes  with  it,  and  the  floating  sec- 
tion may  be  easily  spread  out  into  a  flat  surface.  The 
excess  of  fluid  is  taken  up  with  blotting-paper,  after 
which  a  drop  of  xylol-balsam  is  placed  upon  the  centre 
of  the  section,  and  is  then  covered  with  a  thin,  clean 
cover-slip.  It  is  now  ready  for  examination. 

Each  step  in  the  above  process  has  its  definite  object. 
The  sections  are  placed  in  water  before  staining  in  order 
that  the  diffusion  of  the  straining  solution  into  the  tissues 
may  be  diminished  ;  otherwise  our  efforts  at  rendering 
the  bacteria  more  conspicuous  by  decolorizing  the  tissues 
in  which  they  are  located  would  rob  the  bacteria  of 
their  color  as  well. 

The  acetic  acid  and  also  the  alcohol  are  decolorizers, 
and  are  directed  toward  the  excess  of  staining  in  the 
tissues.  The  cedar  oil  or  xylol  are  bodies  which  mix 
on  the  one  hand  with  alcohol,  on  the  other  with  balsam. 
They  are  known  as  "  clearing  fluids,"  and  not  only  serve 
to  differentiate  the  component  parts  of  the  tissue  but  fill 


STAINING  OF  SECTIONS.  161 

up  the  gap  that  would  otherwise  be  left  in  the  process, 
for  a  section  cannot  be  mounted  in  balsam  directly  from 
alcohol ;  the  two  bodies  do  not  mix  perfectly. 

A  number  of  clearing  agents  are  in  general  use ;  in 
fact,  almost  all  the  essential  oils  come  under  this  head. 
There  is  one — oil  of  cloves — which  is  very  commonly 
used  in  histological  work,  but  it  must  not  be  employed 
in  tissues  containing  bacteria.  It  not  only  extracts  too 
much  color  from  the  bacteria,  but  causes  them  to  fade 
after  the  sections  have  been  mounted  for  a  time. 

When  the  section  thus  stained  and  mounted  is  ex- 
amined microscopically,  it  may  be  found  that  the  tissues 
still  possess  so  much  color  that  the  bacteria  are  not 
visible,  in  which  case  they  have  not  been  decolorized 
sufficiently  ;  or,  on  the  other  hand,  both  bacteria  and 
tissues  may  have  parted  with  their  stains — then  decol- 
orization  has  been  carried  too  far.  In  either  case  the 
fault  must  be  remedied  in  the  manipulation  of  the  next 
section  to  be  mounted. 

In  short,  the  steps  in  the  process  of  staining  sections 
in  general  are  these  : 

a.  From  alcohol  into  distilled  water  for  one  minute. 

b.  Into   the   staining   fluid   for   from   five   to  eight 
minutes. 

c.  Into  water  for  from  three  to  five  minutes. 

d.  Into  0.1   per  cent,  acetic  acid  for  about  one-half 
minute. 

e.  Into  absolute  alcohol  for  a  few  seconds. 

/.  Into  absolute  alcohol  again  for  a  few  seconds. 

g.  Xylol  for  about  one-half  minute. 

h.  Removal  with  spatula  or  section-lifter  to  slide. 

i.  Removal  of  excess  of  xylol. 

'/.  Mounting  in  xylol-balsam. 


162  BACTERIOLOGY. 

The  section  must  be  lifted  from  one  vessel  to  the 
other  by  means  of  either  a  curved  needle  or  a  glass  rod 
drawn  out  to  a  fine  end  and  bent  in  the  form  of  a  curved 
needle. 

By  the  above  process  of  staining,  which  can  be  prac- 
tised as  a  routine  method  for  most  bacteria  in  tissues, 
the  nuclei  of  the  tissue  cells,  as  well  as  the  bacteria,  will 
be  more  or  less  deeply  stained. 

SPECIAL  METHODS  OF  STAINING  BACTERIA  IN 
TISSUES. — For  purposes  of  contrast  stains  it  sometimes 
becomes  necessary  to  completely,  or  nearly  completely, 
decolorize  the  tissues  and  leave  the  bacteria  unaltered 
in  color.  For  this  purpose  special  methods  depending 
on  the  staining  peculiarities  of  the  bacteria  under  con- 
sideration have  been  devised. 

Gram's  method  with  tissues.  One  of  the  most  com- 
monly employed  differential  stains  is  that  of  Gram.  In 
general,  it  is  practised  in  the  way  given  for  its  employ- 
ment on  cover-slip  preparations,  with  some  slight  modi- 
fications. 

In  this  method  the  sections  are  to  be  placed  from 
water  into  a  solution  of  aniline-water  gentian-violet, 
as  prepared  by  the  Koch-Ehrlich  formula,  but  which 
has  been  diluted  with  about  one-third  its  volume  of 
water.  In  this  the  sections  remain  for  about  ten  min- 
utes, preferably  in  a  warm  place,  at  a  temperature  of 
about  40°  C.  They  should  never,  under  any  conditions, 
be  boiled. 

From  this  they  are  washed  alternately  in  the  iodine 
solution  and  alcohol,  occasionally  renewing  the  stained 
with  clean  alcohol,  until  all  color  has  been  extracted 
from  them.  They  are  then  brought  for  one  minute  into 
a  dilute  watery  solution  of  eosin  or  safrauin,  or  Bismarck- 


SPECIAL  METHODS  OF  STAINING  BACTERIA.     163 

brown,  again  washed  out  for  a  few  seconds  in  alcohol, 
and  finally  for  one-fourth  minute  in  absolute  alcohol. 
From  this  they  are  transferred  to  xylol  for  a  half- 
minute.  '  The  remaining  steps  in  the  process  are  the 
same  as  those  given  in  the  general  method.  In  some 
cases  better  results  are  obtained  by  reversing  the  steps 
in  the  process  and  staining  the  bacteria  last,  for  then 
the  frequent  decolorizing  action  of  the  alcohol  on  the 
bacteria  is  diminished ;  thus,  place  the  sections  from 
alcohol  into  eosin,  safranin  or  Bismarck-brown  for  a  few 
minutes,  then  wash  out  in  50  per  cent,  alcohol,  then  for 
from  three  to  five  minutes  in  the  dilute  aniline-water 
gentian-violet  solution,  then  into  the  iodine  bath,  after 
three  minutes  wash  out  in  alcohol,  and,  finally,  for  one- 
fourth  minute  in  absolute  alcohol,  and  then  into  the 
xylol,  from  which  they  may  be  mounted.  Some  of  the 
organisms  which  may  be  stained  by  this  method  are 
micrococcus  tetragenus,  b.  diphtherice,  b.  anthrads,  and 
staph.  pyogenes  aureus.  It  cannot  be  successfully  em- 
ployed with  the  bacillus  of  typhoid  fever. 

Staining  with  dahlia  and  decolorizing  with  sodium  carbo- 
nate solution.  Another  method  that  is  not  very  commonly 
employed,  though  the  results  obtained  by  its  use  are  in 
many  cases  very  satisfactory,  is  to  stain  the  tissues  in  a 
strong  watery  solution  of  dahlia  (about  one-fourth  satu- 
rated) for  from  ten  to  fifteen  minutes ;  from  this  they 
are  transferred  into  a  2  per  cent,  solution  of  sodium  or 
potassium  carbonate,  and  from  this  into  alcohol,  alter- 
nating from  the  one  to  the  other,  until  the  section  is 
almost  colorless.  From  the  alcohol  they  are  rinsed  out 
in  water  and  then  put  into  a  dilute  watery  solution 
of  either  eosin,  Bismarck-brown,  or  safraniu  for  one 
minute,  then  washed  out  in  alcohol,  finally  in  absolute 


164  BACTERIOLOGY. 

alcohol,  and  then  in  xylol,  from  which  they  may  be 
mounted  in  the  manner  given. 

Especially  brilliant  results  are  obtained  when  tissues 
containing  anthrax  bacilli  are  stained  by  this  process ; 
the  bacilli  will  be  of  a  deep  blue  color,  while  the  sur- 
rounding tissues  will  be  of  the  color  used  as  contrast. 

Kuhnds  carbolic  methylene-blue  method.  Stain  the 
sections  in  the  following  solutions  for  from  one-half  to 
one  hour : 

Methylene-blue,  in  substance     .       .       .       .1.5  grammes. 
Absolute  alcohol  .......    10  c.c. 

Rub  up  thoroughly  in  a  mortar,  and  when  the  blue 
is  completely  dissolved,  add  gradually  100  c.c.  of  a  5 
per  cent,  solution  of  carbolic  acid.  (The  solution  de- 
composes after  a  short  time ;  it  should  be  made  fresh 
when  needed.)  From  this  the  sections  are  washed  out 
in  water,  then  in  1.5  to  2  per  cent,  hydrochloric  acid  in 
water,  from  this  they  are  transferred  to  a  solution  of 
lithium  carbonate  of  the  strength  of  six  to  eight  drops 
of  a  concentrated  watery  solution  of  the  salt  to  ten  drops 
of  water,  and  from  this  they  are  again  thoroughly  washed 
in  water,  then  in  absolute  alcohol  containing  enough 
methylene-blue  in  substance  to  give  it  a  tolerably  dense 
color,  then  for  a  few  minutes  in  aniline  oil  to  which  a 
little  methylene-blue  in  substance  has  been  added,  then 
completely  rinse  out  in  pure  aniline  oil,  from  this  they 
are  passed  into  thymol  or  oil  of  turpentine  for  two  min- 
utes, and  then  into  xylol,  from  which  they  are  mounted 
in  xylol-balsam.  The  advantages  of  this  method  are 
that  it  is  generally  applicable,  and  by  its  use  the  bacteria 
are  not  robbed  of  their  color,  whereas  the  tissues  are 
sufficiently  decolorized  to  render  the  bacteria  visible  and 
admit  of  the  use  of  contrast  stains. 


SPECIAL  METHODS  OF  STAINING  BACTERIA.     165 

Weigert's  modification  of  Gram's  method  for  sections. 
Stain  the  sections  in  the  Koch-Ehrlich  aniline-water 
gentian-violet  solution  for  five  or  six  minutes;  wash  out 
in  water  or  physiological  salt  solution  (0.6  to  0.7  per  cent, 
solution  of  sodium  chloride  in  distilled  water) ;  transfer 
them  with  the  section-lifter  to  the  slide ;  take  up  the 
excess  of  fluid  by  gently  pressing  upon  the  flat  section 
with  blotting-paper ;  treat  the  section  with  the  iodine 
solution  used  by  Gram  ;  take  up  the  excess  of  the  solu- 
tion with  blotting-paper;  cover  the  section  with  aniline 
oil — this  not  only  differentiates  the  component  parts  of 
the  section,  but  dehydrates  as  well ;  wash  out  the 
aniline  oil  with  xylol,  and  mount  in  the  usual  way  in 
xylol-balsam.  Or,  decolorization  with  iodine  may  be 
omitted,  and  the  sections,  after  staining  in  the  aniline- 
water  gentian-violet  for  five  or  six  minutes  or  longer,  if 
necessary,  are  transferred  to  the  slide  without  being 
washed  in  water  or  salt  solution,  or  if  so  only  very 
slightly  and  rapidly,  dried  as  completely  as  possible 
with  filter- paper,  then  are  decolorized  with  a  mixture 
of  aniline  oil  (one  part)  and  xylol  (two  parts).  This  is 
the  delicate  part  of  the  process  and  can  be  watched 
under  the  low  power  of  the  microscope.  When  decolor- 
ization is  sufficient  (repeated  applications  of  the  aniline 
oil  and  xylol  mixture  are  generally  necessary),  pure 
xylol  replaces  the  mixture,  and  the  specimen  is  finally 
mounted  in  xylol  balsam.  Unless  all  the  aniline  oil  is 
replaced  by  the  xylol  the  specimen  will  not  keep  well. 
In  this  process  the  aniline  oil  is  really  the  decolorizer 
and  has  the  valuable  property  of  absorbing  a  certain 
amount  of  water,  so  that  dehydration  with  alcohol  is 
avoided.  This  method,  while  it  stains  certain  bacteria 
in  tissues  very  satisfactorily,  is  nevertheless  designed 


166  BACTERIOLOGY. 

especially  for  the  staining  of  fibrin.  Fibrin  and  hya- 
line material  will  be  stained  deep  blue,  bacteria  a  dark 
violet. 

STAINING  OF  TUBERCLE  BACILLI  IN  TISSUES. — As 
for  the  staining  of  cover-slips,  only  those  methods  most 
commonly  employed  will  be  given. 

The  method  of  Ehrlich.  Stain  the  sections  in  aniline- 
water  fuchsin  or  gentian-violet  for  twenty-four  hours ; 
decolorize  in  20  per  cent,  nitric  acid  for  a  few  seconds 
only,  the  color  need  not  be  entirely  extracted  ;  then 
into  70  per  cent,  alcohol  until  no  more  color  can  be  ex- 
tracted by  the  alcohol ;  stain  as  contrast  color  in  dilute 
watery  methylene-blue,  malachite-green,  or  Bismarck- 
brown  solution  ;  wash  out  in  90  per  cent,  alcohol,  then 
in  absolute  alcohol  for  a  few  seconds ;  clear  up  in  xylol 
and  mount  in  xylol-balsam. 

Method  of  Ziehl-Neelsen.  Stain  the  sections  in  warmed 
carbol-fuchsin  solution  for  one  hour ;  temperature  to  be 
about  45°  to  50°  C.  Decolorize  for  a  few  seconds  in  5 
per  cent,  sulphuric  acid,  then  in  70  per  cent,  alcohol, 
and  from  this  on,  as  by  the  Ehrlich  method. 

Dry  method.  For  the  tubercle  bacilli,  as  for  many 
other  organisms  in  tissues,  the  following  method  may 
be  employed  if  only  the  presence  of  organisms  is  to  be 
detected  and  the  histological  condition  of  the  tissues  is 
a  matter  of  no  consequence :  Bring  the  sections  from 
water  upon  a  slide  or  cover-slip,  dry,  fix,  and  stain  by 
the  methods  for  cover-slip  preparations. 

Gray's  method.  The  method  employed  by  Gray  at 
the  Army  Medical  Museum,  Washington,  D.  C.,  a  de- 
scription of  which  is  given  by  Borden,  is  as  follows : 
The  tissue  to  be  stained  should  be  hardened,  preferably 
in  alcohol,  in  pieces  not  exceeding  1.5  by  1.5  by  1  cm. 


STAINING  OF  TUBERCLE  BACILLI  IN  TISSUES.    167 

in  size,  though  tissues  hardened  by  any  other  of  the 
regular  methods  can  be  stained.  Alcohol  is  to  be  pre- 
ferred, however,  as  after  its  use  the  bacilli  stain  more 
quickly  and  brilliantly  than  when  one  of  the  other  hard- 
ening fluids,  Miiller's,  for  instance,  is  employed. 

After  the  tissue  has  been  hardened  it  is  imbedded  in 
paraffin,  and  cut  in  the  usual  manner.  The  sections 
are  then  cemented  to  the  slides  with  a  filtered  J  per 
cent,  solution  of  gold  label  gelatin,  to  which  is  added 
chloral  hydrate  in  the  proportion  of  1  per  cent.,  as 
preservative.  Several  drops  of  this  are  placed  on  a 
slide,  a  section  laid  on  top,  and  the  slide  placed  in  a 
warming  oven,  kept  at  a  temperature  slightly  below  the 
melting  point  of  the  paraffin.  In  about  five  minutes 
all  wrinkles  will  have  been  taken  out  of  the  section, 
which  will  lie  perfectly  flat  and  smooth  on  the  surface 
of  the  gelatin  solution.  The  slide  is  then  removed 
from  the  oven  and  the  surplus  fluid  poured  from  it, 
thus  bringing  the  section  in  contact  with  its  surface, 
after  which  it  is  set  aside  in  a  place  protected  from 
dust,  to  remain  until  the  section  is  firmly  cemented  to 
it  by  the  drying  of  the  gelatin  solution.  The  drying 
may  be  hastened  by  keeping  the  slides  in  an  oven 
below  the  melting-point  of  the  paraffin,  but  it  is  best 
to  set  the  slides  aside  until  the  next  day,  when  the  sec- 
tions will  be  found  to  be  perfectly  cemented  to  them. 
The  paraffin  is  then  removed  from  the  section  by  tur- 
pentine, the  turpentine  by  absolute  alcohol,  the  absolute 
alcohol  by  50  per  cent,  alcohol,  and  this  by  water,  after 
which  the  slides  are  placed  in  a  5  per  cent,  aqueous 
solution  of  potassium  bichromate  for  five  minutes. 
This  renders  the  gelatin  insoluble,  and  prevents  the 
sections  from  leaving  the  slides  during  their  necessarily 


168  BACTERIOLOGY. 

more  or  less  prolonged  immersion  in  the  fuchsin  stain. 
The  potassium  bichromate  is  washed  out  with  water, 
and  the  slides  are  then  placed  in  a  fuchsin  stain,  which 
is  prepared  as  follows : 

Fuchsin 1.5  grammes. 

Absolute  alcohol 14    c.c. 

Carbolic  acid  crystals,  pure    .       .       .       .       .  6    grammes. 

Water 100    c.c. 

Dissolve  the  fuchsin  in  the  alcohol  and  the  carbolic 
acid  in  the  water.  Mix  the  two  solutions  and  let  stand 
for  twelve  hours,  with  occasional  shaking  or  stirring, 
then  filter. 

The  length  of  time  that  the  slide  remains  in  this  solu- 
tion varies  with  circumstances.  The  tubercle  bacilli 
stain  very  quickly ;  in  tissues  properly  hardened  in 
alcohol  five  minutes  is  generally  sufficient  to  stain  them 
them  deeply. 

Prolonged  immersion  in  the  fuchsin  does  no  harm 
and  insures  certainty  of  results.  After  a  section  has 
been  in  the  stain  a  sufficient  length  of  time  it,  with  the 
slide  to  which  it  is  cemented,  is  washed  in  water  until 
the  surplus  stain  is  removed ;  it  is  then  subjected  to 
the  action  of  a  combined  decolorizer  and  contrast  stain 
made  as  follows : 

Methyl-blue 2. 25  grammes. 

Absolute  alcohol 30    c.c. 

Sulphuric  acid 12      " 

Water  (distilled) 100      " 

Dissolve  the  methyl-blue  in  the  alcohol,  add  the  acid 
to  the  water,  mix  the  two  solutions,  and  let  stand,  with 
occasional  shaking,  for  twelve  hours,  then  filter. 

This  solution  is  allowed  to  act  upon  the  tissue  for  a 
few  seconds,  and  as  soon  as  the  blue  color  predominates 
over  the  red,  as  seen  by  transmitted  light,  the  section  is 


STAINING  OF  TUBERCLE  BACILLI  IN  TISSUES.     169 

immediately  washed  in  water.  Generally  the  red  color 
reappears  and  the  section  must  be  again  subjected  to  the 
action  of  the  blue  solution  and  again  washed  in  water. 
This  must  be  repeated  until  the  blue  almost,  if  not  quite 
completely  and  permanently,  replaces  the  red  stain.  This 
is  the  most  important  part  of  the  process  and  entirely 
satisfactory  results  are  only  obtained  after  some  practice. 
The  tendency  is  usually  to  not  sufficiently  replace  the 
fuchsin  with  the  methyl-blue,  and  in  consequence  the 
red  color  of  the  bacilli  is  masked  by  the  red  of  the 
surrounding  tissues.  Unless  all  acid  is  thoroughly 
removed  by  the  final  washing  in  water  the  stain  is  not 
permanent.  The  section  is  then  completely  dehydrated 
with  absolute  alcohol,  after  taking  up  the  excess  of 
water  on  the  slide  with  blotting-paper.  The  alcohol  is 
followed  by  turpentine,  and  the  process  is  completed  by 
mounting  in  xylol  balsam. 

In  case  it  is  desired  to  stain  sections  cut  by  the 
freezing  method,  they  are  placed  upon  a  slide  on  which 
a  few  drops  of  the  gelatin  fixative  have  been  placed,  and 
after  about  five  minutes,  during  which  the  fixative  will 
have  penetrated  the  section,  the  surplus  is  poured  from 
beneath  the  section.  The  slides  are  then  set  aside  for 
the  gelatin  to  harden  by  drying,  and  after  drying  they 
are  placed  in  bichromate  fluid  to  render  the  gelatin 
insoluble.  They  are  then  manipulated  in  exactly  the 
same  manner  as  the  sections  cut  by  the  paraffin  method. 

This  method  gives  equally  good  results  with  tissues 
containing  the  lepra  bacillus  as  with  those  containing 
tubercle  bacilli. 


CHAPTER  XL 

Systematic  study  of  an  organism— Points  to  be  considered  in  identifying 
an  organism  as  a  definite  species. 

AFTER  isolating  an  organism  by  the  plate  method, 
considerable  work  is  necessary  in  order  to  establish  its 
identity  as  a  definite  species. 

It  must  possess  certain  morphological  and  cultural 
peculiarities,  which  must  be  constant  under  constant 
conditions. 

Its  form  at  different  stages  must  always  be  the  same. 
Its  ability  or  inability  to  produce  spores  must  not  vary 
under  proper  conditions.  Its  growth  upon  the  different 
media  under  constant  conditions  of  temperature  and 
reaction  must  always  present  the  same  outward  appear- 
ances. The  changes  brought  about  by  it  in  the  reaction 
of  the  media  in  which  it  is  growing  must  follow  a  fixed 
rule.  Its  power  to  produce  liquefaction  of  the  gelatin, 
or  to  grow  upon  it  without  bringing  about  this  change, 
must  always  be  the  same.  Its  motility  or  non-motility, 
and,  if  motile,  the  approximate  number  and  position  of 
its  organs  of  locomotion,  must  be  determined.  Its  pro- 
duction of  certain  chemical  products  must  be  detected 
by  chemical  analysis.  Its  behavior  toward  oxygen — 
i.  e.,  does  it  require  this  gas  for  its  growth  ?  is  this  gas 
an  indifferent  factor  ?  or  by  its  presence  are  the  life  pro- 
cesses of  the  organism  checked? — must  be  decided.  Its 
behavior  under  varying  conditions  of  temperature  and 
under  the  influence  of  different  chemical  bodies,  as  well 


DIFFERENT  PARTS  OF  THE  MICROSCOPE.    171 

as  its  growth  in  media  of  different  reactions,  are  to  be 
studied.  The  property  of  producing  fermentation  with 
the  liberation  of  gases,  and  the  character  and  quantita- 
tive relations  of  these  gases  must  be  ascertained ;  if  it 
produces  pigment,  what  are  the  conditions  favorable  and 
unfavorable  to  this  function  ;  and,  lastly,  we  must  con- 
sider its  behavior  when  introduced  into  the  bodies  of 
animals  used  for  experimental  work — i.  e.,  is  it  a  dis- 
ease-producing organism,  or  does  it  belong  to  the  group 
of  innocent  saprophytes? 

We  have  learned  the  methods  of  obtaining  colonies, 
and  have  acquainted  ourselves  with  some  of  the  pecu- 
liarities by  which  they  are  distinguished  from  one  an- 
other. The  next  important  step  is  to  determine  the 
morphology  of  the  individuals  composing  these  colonies 
as  well  as  their  relation  to  each  other  in  the  colony. 
These  points  are  decided  by  microscopic  examination 
of  bits  of  the  colony  which  are  transferred  to  thin  glass 
cover-slips,  upon  which  they  are  dried,  stained,  and 
mounted.  Cover-slips  for  this  purpose  are  prepared  in 
two  ways  :  either  by  taking  up  a  bit  of  the  colony  on  a 
platinum  needle,  smearing  it  upon  a  cover-slip,  staining 
it,  and  examining  it — by  which  only  the  morphology  of 
the  individual  bacteria  can  be  made  out — or  by  the  method 
of  u  impression  cover-slip  preparations,"  by  which  not 
only  the  morphology,  but  also  the  relation  of  the  organ- 
isms to  one  another  in  the  colony  can  be  determined.  The 
details  of  these  methods  will  be  found  in  the  chapter  on 
the  method  of  staining. 

MICROSCOPIC  EXAMINATION  OF  PREPARATIONS. 

THE  DIFFERENT  PARTS  OF  THE  MICROSCOPE. — 
Before  describing  the  process  of  examining  preparations 


172  BA  CTERIOL  OGY. 

microscopically,  a  few  definitions  of  the  terms  used  in 
referring  to  the  microscope  may  not  be  out  of  place. 

The  ocular  or  eye-piece  is  the  lens  at  which  the  eye  is 
placed  in  looking  through  the  instrument.  It  serves  to 
magnify  the  image  projected  through  the  objective. 

The  objective  is  the  lens  which  is  at  the  distal  end  of 
the  barrel  of  the  instrument,  and  which  serves  to  mag- 
nify the  object  to  be  examined. 

The  stage  is  the  shelf  or  platform  of  the  microscope 
on  which  the  object  rests. 

The  diaphragms  are  the  perforated  stops  that  fit  in  the 
centre  of  the  stage.  They  vary  in  size  so  that  different 
amounts  of  light  may  be  admitted  to  the  object  by  using 
diaphragms  with  larger  or  smaller  openings. 

The  "  Iris  "  diaphragm  opens  and  closes  like  the  iris  of 
the  eye.  It  is  so  arranged  that  its  opening  for  admission 
of  light  can  be  increased  or  diminished  by  moving  a 
small  arm  in  one  or  another  direction. 

The  reflector  is  the  mirror  placed  beneath  the  stage, 
which  serves  to  direct  the  light  to  the  object  to  be  exam- 
ined. 

The  coarse  adjustment  is  the  rack-and-pinion  arrange- 
ment by  which  the  barrel  of  the  microscope  can  be  quickly 
raised  or  lowered. 

The  fine  adjustment  serves  to  raise  and  lower  the  barrel 
of  the  instrument  very  slowly  and  gradually. 

For  the  microscopic  study  of  bacteria  it  is  essential 
that  the  microscope  be  provided  with  an  oil-immersion 
system  and  a  sub-stage  condensing  apparatus. 

The  oil-immersion  or  Homogeneous  System  consists  in  an 
objective  so  constructed  that  it  can  only  be  used  when  the 
transparent  media  through  which  the  light  passes  in  enter- 
ing it  are  all  of  the  same  index  of  refraction — i.  e.,  are 


EXAMINATION  OF  COVER-SLIPS.  173 

homogeneous.  This  is  accomplished  by  interposing  be- 
tween the  face  of  the  lens  and  the  cover-slip  covering 
the  object  to  be  examined,  a  body  which  refracts  the 
light  in  the  same  way  as  do  the  glass  slide,  the  cover- 
slip,  and  the  glass  of  which  the  objective  is  made.  For 
this  purpose  a  drop  of  oil  of  the  same  index  of  refrac- 
tion as  the  glass  is  placed  upon  the  face  of  the  lens,  and 
the  examinations  are  made  through  this  oil.  There  is 
thus  no  loss  of  light  from  deflection,  as  is  the  case  in 
the  dry  system. 

The  sub-stage  condensing  apparatus  is  a  system  of 
lenses  situated  beneath  the  central  opening  of  the  stage. 
They  serve  to  condense  the  light  passing  from  the 
reflector  to  the  object  in  such  a  way  that  it  is  focussed 
upon  the  object,  thus  furnishing  the  greatest  amount  of 
illumination.  Between  the  condenser  and  reflector  is 
placed  an  adjustable  diaphragm,  the  aperture  of  which 
can  be  regulated,  as  circumstances  require,  to  permit  of 
either  a  very  small  or  very  large  amount  of  light  pass- 
ing to  the  object. 

MICROSCOPIC  EXAMINATION  OF  COVER-SLIPS. — 
The  stained  cover-slip  is  to  be  examined  with  the  oil- 
immersiou  objective,  and  with  the  diaphragm  of  the 
sub-sta^e  condensing  apparatus  open  to  its  full  extent. 
The  object  gained  by  allowing  the  light  to  enter  in  such 
a  large  volume  is  that  the  contrast  produced  by  the 
colored  bacteria  in  the  brightly  illuminated  field  is 
much  more  conspicuous  than  when  a  smaller  amount  of 
light  is  thrown  upon  them.  This  is  true  not  only  for 
stained  bacteria  on  cover-slips,  but  likewise  for  their 
differentiation  from  surrounding  objects  when  they  are 
located  in  tissues.  With  unstained  bacteria  and  tissues, 
on  the  contrary,  the  structure  can  best  be  made  out 


174  BACTERIOLOGY. 

by  reducing  the  bundle  of  light-rays  to  its  smallest 
amount  compatible  with  distinct  vision,  and  in  this  way 
favoring,  not  color  contrast,  but  contrasts  which  appear 
as  lights  and  shadows,  due  to  the  differences  in  perme- 
ability to  light  of  the  various  parts  of  the  material  under 
examination. 

STEPS  IN  EXAMINING  STAINED  PREPARATIONS 
WITH  THE  OIL-IMMERSION  SYSTEM. — Place  upon  the 
centre  of  the  cover-slip  which  covers  the  preparation  a 
small  drop  of  immersion  oil.  Place  the  slide  upon  the 
centre  of  the  stage  of  the  microscope.  With  the  coarse 
adjustment  lower  the  oil-immersion  objective  until  it 
just  touches  the  drop  of  oil.  Open  the  illuminating 
apparatus  to  its  full  extent.  Then,  with  the  eye  to 
the  ocular  and  the  hand  on  the  fine  adjustment,  turn  the 
adjusting-screw  toward  the  right  until  the  field  becomes 
somewhat  colored  in  appearance.  When  this  is  seen,  pro- 
ceed more  slowly  in  the  same  direction,  and,  after  one  or 
two  turns,  the  object  will  be  in  focus.  Do  not  remove  the 
eye  from  the  instrument  until  this  has  been  accomplished. 

Then,  with  one  hand  upon  the  fine  adjustment  and 
the  thumb  and  index  finger  of  the  other  hand  holding 
the  slide  lightly  by  its  end,  the  slide  may  be  moved 
about  under  the  objective.  At  the  same  time  the  screw 
of  the  fine  adjustment  must  be  turned  back  and  forth  so 
that  the  different  levels  of  the  preparation  may  one  after 
the  other  be  brought  into  focus.  In  this  way  the  whole 
section  or  preparation  may  be  inspected.  When  the 
examination  is  finished,  raise  the  objective  from  the 
preparation  by  turning  the  screw  of  the  coarse  adjust- 
ment toward  you.  Remove  the  preparation  from  the 
stage,  and,  with  a  fiue  silk  cloth  or  handkerchief,  wipe 
very  gently  and  carefully  the  oil  from  the  face  of  the  lens. 


EXAMINATION  OF  UNSTAINED  PREPARATIONS.     175 

The  lens  is  then  unscrewed  from  the  microscope  and 
placed  in  the  case  intended  for  its  reception. 

During  work,  of  course,  the  lens  need  not  be  cleaned 
and  put  away  after  each  examination ;  but  when  the 
work  for  the  day  is  over,  an  immersion  lens  must  always 
be  protected  in  this  way.  Under  no  circumstances  should 
it  be  allowed  to  remain  in  the  immersion  oil  or  exposed 
to  dust  for  any  length  of  time. 

EXAMINATION  OF  UNSTAINED  PREPARATIONS. — 
''Hanging  drops"  It  frequently  becomes  necessary  to 
examine  bacteria  in  the  unstained  condition.  The  cir- 
cumstances calling  for  this  arise  while  studying  the 
multiplication  of  cells,  the  germination  of  spores,  the 
formation  of  spores,  and  the  absence  or  presence  of 
motility. 

In  this  method  the  organisms  to  be  studied  are  sus- 
pended in  a  drop  of  physiological  salt  solution  or  bouillon 
in  the  centre  of  a  cover-slip.  This  is  then  placed,  drop 
down,  upon  a  slide  in  the  centre  of  which  a  hollow  or 
depression  is  ground  (Fig.  33).  The  slip  is  held  in  posi- 
tion by  a  thin  layer  of  vaselin,  which  may  be  painted 

FIG.  33. 


Hollow-ground  glass  slide  for  observing  bacteria  in  hanging  drops. 

around  the  margins  of  the  depression.  This  not  only 
prevents  the  slip  from  moving  from  its  position  during 
examination,  but  also  prevents  drying  by  evaporation  if 
the  preparation  is  to  be  observed  for  any  length  of 
time.  This  is  known  as  the  " hanging-drop"  method  of 
examination  or  cultivation.  It  is  indispensable  for  the 
purposes  mentioned,  and  at  the  same  times  requires  con- 


176  BACTERIOLOGY. 

siderable  care  in  its  manipulation.  The  fluid  is  so  trans- 
parent that  the  cover-slip  is  often  broken  and  the  face 
of  the  objective  injured  by  its  being  brought  down  upon 
the  preparation  before  one  is  aware  that  the  focal  dis- 
tance has  been  reached.  This  may  be  avoided  by  grasp- 
ing the  slide  with  the  left  hand  and  moving  it  back 
and  forth  under  the  objective  as  it  is  brought  down 
toward  the  object.  As  soon  as  the  least  pressure  is  felt 
upon  the  slide  the  objective  must  be  raised,  otherwise 
the  cover-slip  will  be  broken  and  the  lens  may  be  ren- 
dered worthless. 

A  safer  plan  is  to  bring  the  edge  of  the  drop  into  the 
centre  of  the  field  with  one  of  the  higher  power  dry 
lenses.  When  this  is  accomplished,  substitute  the  im- 
mersion for  the  dry  system,  and  the  edge  of  the  drop 
should  now  be  somewhere  near  the  centre  of  the  field. 

In  examining  bacteria  by  this  method  there  is  a  pos- 
sibility of  error  that  must  be  guarded  against.  All 
microscopic  insoluble  particles  in  suspension  in  fluids 
possess  a  peculiar  tremor  or  vibratory  motion,  the  so- 
called  "  Browuian  motion. "  This  is  very  apt  to  give 
the  impression  that  the  organisms  under  examination 
are  motile,  when  in  truth  they  are  not  so,  their  move- 
ment in  the  fluid  being  due  only  to  this  molecular 
tremor. 

The  difference  between  the  motion  of  bodies  under- 
going this  molecular  tremor  and  that  possessed  by  cer- 
tain living  bacteria  is  that  the  former  particles  never 
move  from  their  place  in  the  field,  while  the  living 
bacteria  alter  their  position  in  relation  to  the  surround- 
ing organisms,  and  may  dart  from  one  position  in  the 
field  to  another.  With  some  cases  the  true  movement 
of  bacteria  is  very  slow  and  undulating,  while  in  others 


SPORE-FORMATION.  177 

it  is  rapid  and  darting.     The  molecular  tremor  may  be 
seen  with  non-motile  and  with  dead  organisms. 

NOTE. — Prepare  three  hanging-drop  preparations — 
one  from  a  drop  of  dilute  India-ink,  a  second  from  a 
culture  of  micrococci,  and  a  third  from  a  culture  of  the 
bacillus  of  typhoid  fever.  In  what  way  do  they  differ? 

STUDY  OF  SPOKE-FORMATION. — The  hanging-drop 
method  just  mentioned  is  not  only  employed  for  detect- 
ing the  motility  of  an  organism,  but  also  for  the  study  of 
its  spore-forming  properties. 

Since  with  aerobic  organisms  spore-formation  occurs, 
as  a  rule,  only  in  the  presence  of  oxygen,  and  is  induced 
more  by  limitation  of  the  nutrition  of  the  organisms 
than  by  any  other  factor,  it  is  essential  that  these  two 
points  should  be  borne  in  mind  in  preparing  the  drop 
cultures  in  which  the  process  is  to  be  studied.  For  this 
reason  the  drop  of  bouillon  should  be  small  and  the  air- 
chamber  relatively  large. 

The  cover-slip  and  hollow-ground  slide  should  be 
carefully  sterilized,  and  with  a  sterilized  platinum  loop 
a  very  small  drop  of  bouillon  is  placed  in  the  centre  of 
the  cover-slip,  The  slip  is  then  inverted  over  the  hol- 
low depression  in  the  sterilized  object-glass  and  sealed 
with  vaselin.  The  most  convenient  method  of  perform- 
ing this  last  step  in  the  process  is  to  paint  a  ring  of 
vaselin  around  the  edges  of  the  hollow  in  the  slide,  and 
then,  without  taking  the  cover-slip  up  from  the  table 
upon  which  it  rests,  invert  the  hollow  over  the  drop  and 
press  it  gently  down  upon  the  cover-slip.  The  vaseliu 
causes  the  slip  to  adhere  to  the  slide,  so  that  it  can  be 
easily  taken  up.  The  drop  now  hangs  in  the  centre  of 


178  BACTERIOLOGY. 

the  small  air-tight  chamber  which  exists  between  the 
depression  in  the  slide  and  the  cover-slip.  (See  Fig.  32, 
page  157.) 

A  very  thin  drop  of  sterilized  agar-agar  may  be  sub- 
stituted for  the  bouillon.  It  serves  to  retain  the  organ- 
isms in  a  fixed  position,  and  the  process  may  be  more 
easily  followed. 

As  soon  as  finished,  the  preparation  is  to  be  examined 
microscopically,  and  the  condition  of  the  organisms 
noted.  It  is  then  to  be  retained  in  a  warm  chamber 
especially  devised  for  the  purpose,  and  kept  under  con- 
tinuous observation.  The  form  of  chamber  best  adapted 
for  the  purpose  is  one  which  envelops  the  whole  micro- 
scope. It  is  provided  with  a  window  through  which 
the  light  enters,  and  an  arrangement  for  moving  the 
slide  about  from  the  outside.  The  formation  of  spores 
requires  a  much  longer  time  than  the  germination  of 
spores  into  bacilli,  but  with  patience  both  processes  may 
be  satisfactorily  observed. 

It  will  be  noticed  that  the  description  of  this  process 
is  very  much  like  that  which  has  just  been  given,  but 
differs  from  it  in  one  respect,  viz.,  that  in  this  manipu- 
lation we  are  not  making  a  preparation  which  is  simply 
to  be  examined  and  then  thrown  aside,  but  it  is  an  actual 
pure  culture,  and  must  be  kept  as  such,  otherwise  the 
observation  will  be  worthless.  For  this  reason  the 
greatest  care  must  be  observed  in  the  sterilization  of  all 
objects  employed.  Studies  upon  spore- formation  by  this 
method  frequently  continue  over  hours,  and  sometimes 
days,  and  contamination  must,  therefore,  be  carefully 
guarded  against.  The  study  should  be  begun  with  the 
vegetative  form  of  the  organisms;  the  hanging-drop 
preparation  should,  for  this  reason,  always  be  made 


GELATIN  CULTURES.  179 

from  a  perfectly  fresh  culture  of  the  organism  under 
consideration,  before  time  has  elapsed  for  spores  to 
form. 

The  simple  detection  of  the  presence  or  absence  of 
spore-formation  can  in  many  cases  be  made  by  other 
methods.  For  example,  many  species  of  bacteria  which 
possess  this  property  form  spores  most  readily  upon 
media  from  which  it  is  somewhat  difficult  for  them  to 
obtain  the  necessary  nutrition ;  potatoes  and  agar-agar 
that  have  become  a  little  dry  offer  very  favorable  con- 
ditions, because  of  the  limited  area  from  which  the 
growing  bacteria  can  draw  their  nutritive  supplies  and 
because  of  the  free  access  which  they  have  to  oxygen  ; 
for,  their  growth  being  on  the  surface,  they  are  sur- 
rounded by  this  gas  unless  means  are  taken  to  prevent 
it.  By  the  hanging-drop  method,  however,  more  than 
this  simple  property  may  be  determined.  It  is  possible 
not  only  to  detect  the  stages  and  steps  in  the  formation 
of  endogenous  spores,  but  when  the  spores  are  completely 
formed  by  transferring  them  to  a  fresh  bouillon-drop  or 
drop  of  agar-agar,  preserved  in  the  same  way,  their  ger- 
mination into  mature  rods  may  be  seen.  The  word 
rods  is  used  because  as  yet  we  have  no  evidence  that 
endogenous  spore-formation  occurs  in  any  of  the  other 
morphological  groups  of  bacteria. 

STUDY  OF  GELATIN  CULTURES. — As  has  been  pre- 
viously stated,  the  behavior  of  bacteria  toward  gelatin 
differs — some  of  them  producing  apparently  no  altera- 
tion in  the  medium,  while  others  bring  about  a  form  of 
peptonization  which  results  in  liquefaction  of  the  gela- 
tin at  and  around  the  place  at  which  the  colonies  are 
growing.  In  some  instances  this  liquefaction  spreads 
laterally  and  downward,  causing  a  saucer-shaped  exca- 


180  BACTERIOLOGY. 

vation,  while  in  others  the  colony  sinks  directly  down 
into  the  gelatin  and  may  be  seen  lying  at  the  bottom  of 
a  funnel-shaped  depression.  These  differences  are  con- 
stantly employed  as  one  of  the  means  of  differentiating 
otherwise  closely  allied  members  of  the  same  family  of 
bacteria.  Studies  upon  the  spirillum  of  Asiatic  cholera 
and  a  number  of  closely  allied  species,  for  example, 
reveal  a  decided  difference  in  the  form  of  liquefaction 
produced  by  these  different  organisms.  The  slightest 
detail  in  this  respect  must  be  noted,  and  its  frequency 
or  constancy  under  different  conditions  determined. 

CULTURES  ON  POTATO. — A  very  important  feature 
in  the  study  of  an  organism  is  its  growth  on  sterilized 
potato.  Many  organisms  present  appearances  under 
this  method  of  cultivation  which  alone  can  almost  be 
considered  characteristic.  In  some  cases  coarsely  lobu- 
lated,  elevated,  dry  or  moist  patches  of  development 
occur  after  a  few  hours ;  again,  the  growth  may  be  finely 
granular  and  but  slightly  elevated  above  the  surface  of 
the  potato ;  at  one  time  it  will  be  dry  and  dull  in  ap- 
pearance, again  it  may  be  moist  and  glistening.  Some- 
times there  is  a  production  of  bubbles,  owing  to  fermen- 
tation brought  about  by  the  growth  of  the  organs. 

A  most  striking  form  of  development  on  potato  is 
that  possessed  by  the  bacillus  of  typhoid  fever  and  the 
bacillus  of  diphtheria.  After  the  inoculation  of  a  potato 
with  either  of  these  organisms  there  is  usually  no  naked- 
eye  evidence  of  a  growth  in  either  instance,  though  micro- 
scopic examination  of  scrapings  from  the  surface  of  the 
potato  reveals  an  active  multiplication  of  the  organisms 
which  had  been  planted  there.  The  potato  is  one  of 
the  most  important  differential  media  which  we  possess 
for  this  work. 


ANILINE  D  YES  FOR  DIFFERENTIAL  DIA  GNOSIS.    181 

REACTION    PRODUCED   BY    ORGANISMS  IN  THEIR 

ROWTH. — The  reactions  produced  in  the  media  by 
different  organisms  in  the  course  of  their  growth  are 
very  valuable  as  means  of  differentiation. 

In  some  cases  these  changes  are  so  marked  that  they 
are  readily  detected  by  the  coarser  reagents ;  again  they 
are  so  slight  as  to  require  the  employment  of  the  most 
delicate  indicators.  They  are  sometimes  seen  to  produce 
at  one  period  of  their  growth  an  alkaline,  at  another 
period  an  acid  reaction.  This  is  seen  in  the  cultures  of 
bacillus  diphtherice  of  Loeffler. 

These  differences  are  best  seen  after  the  addition  to  the 
media  in  which  the  organisms  are  to  grow,  of  some  of 
the  chemical  substances  which  do  not  interfere  with  the 
development  of  the  organisms,  but  which  under  one 
reaction  are  of  one  color,  and  with  an  alteration  of  the 
reaction  become  a  different  color,  the  change  being  in- 
dicated by  the  play  of  colors.  Such  substances  as  litmus 
in  the  form  of  the  so-called  "  litmus  tincture,"  and 
coralline  (rosolic  acid)  in  alcoholic  solution  have  been 
employed  for  this  purpose.  They  may  be  added  to  the 
media  in  the  proportions  given  in  the  chapter  on  media, 
and  the  alterations  in  their  colors  studied  with  different 
bacteria.  Milk  and  litmus  tincture  or  peptone  solution 
to  which  rosolic  acid  has  been  added  are  very  favorable 
media  for  this  experiment. 

In  milk,  coagula  will  now  and  then  appear  as  a  result 
of  acids  produced  during  the  bacterial  life,  while  again 
acids  maybe  produced  and  yet  no  coagulation  be  noticed. 

ANILINE  DYES  FOR  DIFFERENTIAL  DIAGNOSIS  — 
The  addition  to  solid  media  of  some  of  the  aniline  dyes, 
fuchsin,  methylene-blue,  methylene-green,  and  several 
others,  as  well  as  combinations  of  these  dyes,  have  been 

9 


182  BACTERIOLOGY. 

recommended  as  a  means  of  differentiation  of  organisms, 
the  differences  claimed  to  be  produced  consisting  of 
alterations  in  the  color  of  the  media  due  to  oxidizing  or 
reducing  properties  of  the  growing  bacteria.  As  yet 
but  little  has  come  from  this  method  of  work.  It  can- 
not at  present  be  recommended  as  a  reliable  means  of 
diagnosis. 

BEHAVIOR  TOWARD  STAINING  REAGENTS.  —  The 
behavior  of  certain  organisms  toward  the  different  dyes 
and  their  reactions  under  special  methods  of  after- 
treatment  serve  as  aids  to  their  diagnosis.  With  very 
few  exceptions  bacteria  stain  readily  with  the  common 
aniline  dyes,  but  they  differ  materially  in  the  tenacity 
with  which  they  retain  these  colors  under  the  subsequent 
treatment  with  decolorizing  agents. 

The  tubercle  bacillus  and  the  bacillus  of  leprosy,  for 
example,  are  difficult  to  stain,  but  when  once  stained 
retain  their  color  under  the  action  of  such  energetic 
decolorizing  agents  as  alcohol,  nitric  acid,  oxalic 
acid,  etc. 

Certain  other  organisms  when  stained  with  a  solution 
of  gentian  violet  in  aniline-water,  retain  their  color  when 
treated  with  such  decolorizing  bodies  as  iodine  solution 
and  alcohol  (Gram's  method),  while  again  others  are 
completely  decolorized  by  this  method. 

Many  of  them  can  only  be  treated  with  water,  or  but 
for  a  few  seconds  with  alcohol,  without  losing  their 
color. 

It  is  essential  that  these  peculiarities  should  be  care- 
fully noted  in  studying  an  organism. 

FERMENTATION. — The  production  of  gas  as  an  in- 
dication of  fermentation  is  an  accompaniment  of  the 
growth  of  some  organisms.  This  is  best  studied  in 


FEEMEN1ATION.  183 

media  to  which  1  to  2  per  cent,  of  grape  sugar  (glucose) 
has  been  added. 

In  this  experiment  the  test-tube  should  be  filled  to 
about  one-half  its  volume  with  agar-agar.  The  medium 
is  then  liquefied,  and  when  at  the  proper  temperature, 
a  small  quantity  of  a  pure  culture  of  the  organism  under 
consideration  should  be  carefully  distributed  through 
it.  The  tube  is  then  placed  in  ice-water  and  rapidly 
solidified  in  the  vertical  position.  When  solid  it  is 
placed  in  the  incubator.  After  twenty-four  to  thirty- 
six  hours,  if  the  organism  possesses  the  property  of 
causing  fermentation  of  sugar,  the  medium  will  be  dotted 
everywhere  with  very  small  cavities  containing  the  gas 
that  has  resulted. 

This  property  of  fermentation  with  production  of  gas 
is  of  such  importance  as  a  differential  means  that  latterly 
considerable  attention  has  been  given  to  it,  and  those 
who  have  been  most  intimately  concerned  in  the  devel- 
opment of  our  knowledge  on  the  subject  do  not  consider 
it  enough  to  say  that  the  growth  of  an  organism  "is 
accompanied  by  the  production  of  gas- bubbles,"  but  that 
under  given  conditions  we  should  determine  not  only  the 
amount  of  gas  or  gases  produced  by  the  organism  under 
consideration  but  also  their  nature  and  quality.  For  this 
purpose  Smith1  recommends  the  employment  of  the  fer- 
mentation-tube used  by  Einhorn  in  the  quantitative  fer- 
mentation test  for  sugar  in  the  urine.  It  is  a  tube  bent 
at  an  acute  angle,  closed  at  one  end  and  enlarged  with  a 
bulb  at  the  other.  At  the  bend  the  tube  is  constricted. 
To  it  a  glass  foot  is  attached  so  that  the  tube  may  stand 

1  An  excellent  and  exhaustive  contribution  to  this  subject  has  been  made 
by  Theobald  Smith  in  "  The  Wilder  Quarter-Century  Book,"  Ithaca,  N.  Y., 


184 


BACTERIOLOGY. 


FIG.  34. 


upright.  (See  Fig.  34.)  To  fill  the  tube  the  fluid 
(they  are  only  used  with  fluid  media)  is  poured  into  the 
bulb  until  this  is  about  half  full.  The  tube  is  then 
tilted  until  the  closed  arm  is  nearly  horizontal,  so  that 
the  air  may  flow  out  into  the  bulb  and  the  fluid  flow 
into  the  closed  arm  to  take  its  place.  When  this  has 
been  completely  filled  enough  fluid  should  be  added  to 
cover  the  lowest  expanding  portion  of  the  bulb,  and  the 
opening  of  the  bulb  plugged  with  cotton.  They  are 
then  to  be  sterilized.  During  sterilization  they  are  to 
be  maintained  in  the  upright  position.  Under  the  in- 
fluence of  heat  the  tension  of  water- 
vapor  in  the  closed  arm  forces  most 
of  the  fluid  into  the  bulb.  As  the 
tubes  cool  the  fluid  returns  to  its 
place  in  the  closed  arm  and  fills  it 
again  with  the  exception  of  a  small 
space  at  the  top  which  is  occupied  by 
the  air  originally  dissolved  in  the 
liquid  and  which  has  been  driven 
out  by  the  heat.  The  air-bubble 
should  be  tilted  out  after  each  ster- 
ilization, and  finally,  after  the  third 
exposure  to  steam  this  arm  of  the 
tube  will  be  free  from  air. 

The  medium  employed  is  bouillon 
containing  some  fermentable   carbo- 
's  fermentation-  hydrate,  as  glucose,  lactose,  or  sac- 
charose.     After  inoculation  the  flasks 
are  placed  in  the  incubator  and  the  amount  of  gas  that 
collects  in  the  closed  arm  is,  from  day  to  day,  noted. 

From  studies  that  have  been  made  this  gas  is  found  to 
consist  usually  of  about  one  part  by  volume  of  carbonic 


CULTIVATION  WITHOUT  OXYGEN.  185 

acid  and  two  parts  by  volume  of  an  explosive  gas  consist- 
ing largely  of  hydrogen.  For  determining  the  nature  and 
quantitative  relations  of  these  gases  Smith1  recommends 
the  following  procedure  :  "  The  bulb  is  completely  filled 
with  a  2  per  cent,  solution  of  sodium  hydroxide  (NaOH) 
and  closed  tightly  with  the  thumb.  The  fluid  is  shaken 
thoroughly  with  the  gas  and  allowed  to  flow  back  and 
forth  from  bulb  to  closed  branch  and  the  reverse  several 
times  to  insure  intimate  contact  of  the  CO2  with  the 
alkali.  Lastly,  before  removing  the  thumb  all  the  gas  is 
allowed  to  collect  in  the  closed  branch,  so  that  none  may 
escape  when  the  thumb  is  removed.  If  CO2  be  present 
a  partial  vacuum  in  the  closed  branch  causes  the  fluid  to 
rise  suddenly  when  the  thumb  is  removed.  After 
allowing  the  layer  of  foam  to  subside  somewhat  the 
space  occupied  by  gas  is  again  measured,  and  the  dif- 
ference between  this  amount  and  that  measured  before 
shaking  with  the  sodium  hydroxide  solution  gives  the 
proportion  of  CO2  absorbed.  The  explosive  character 
of  the  residue  is  determined  as  follows  :  The  cotton  plug 
is  replaced  and  the  gas  from  the  closed  branch  is  allowed 
to  flow  into  the  bulb  and  mix  with  the  air  there  present. 
The  plug  is  then  removed  and  a  lighted  match  inserted 
into  the  mouth  of  the  bulb.  The  intensity  of  the  ex- 
plosion varies  with  the  amount  of  air  present  in  the 
bulb." 

CULTIVATION  WITHOUT  OXYGEN. — As  we  have 
already  learned,  there  is  a  group  of  organisms  to  which 
the  name  "  anaerobic  organisms  "  has  been  given,  which 
are  characterized  by  their  inability  to  grow  in  the  pres- 
ence of  oxygen.  For  the  cultivation  of  the  members  of 

i  Loc.  cit.,  p.  196. 


186 


BACTERIOLOGY. 


FIG.  35. 


this  group  a  number  of  devices  are  employed  for  the 
exclusion  of  oxygen  from  the  cultures. 

Koch's  method.  Koch  covered  the  surface  of  a  gel- 
atin plate,  which  had  been  previously  inoculated,  with  a 
thin  sheet  of  sterilized  isinglass.  The  organisms  which 
grew  beneath  it  were  supposed  to  grow  without  oxygen. 
Hesse's  method.  Hesse  poured  sterilized  oil  upon  the 
surface  of  a  culture  made  by  stabbing  into  a  tube  of 
gelatin.  The  growth  that  occurred  along  the  track  of 
the  needle  was  supposed  to  be  anaerobic  in  nature. 

Methods  of  Liborius.  Liborius  has  suggested  two 
useful  methods  for  this  purpose.  The  one  is  to  fill  a 
test-tube  about  three-quarters 
full  of  gelatin  or  agar-agar, 
which,  after  having  been  ster- 
ilized, is  to  be  kept  in  a  vessel 
of  boiling  water  for  ten  min- 
utes to  expel  all  air  from  it. 
It  is  then  rapidly  cooled  in 
ice-water,  and  when  between 
30°  and  40°  C.,  still  fluid,  is 
to  be  inoculated  and  very  rap- 
idly solidified.  It  is  then 
sealed  up  in  the  flame.  An- 
aerobic bacteria  develop  only 
in  the  lower  layers  of  the 
medium.  His  other  method  is 
that  in  which  he  employs  a 
special  tube,  known  as  "the 
Liborius  tube."  Its  construc- 
tion is  shown  in  Fig.  35. 


Liborius's  tube  for  anaerobic 
cultures. 


Through  the  side  tube  hydrogen  is  passed  until  all  air 
is  expelled ;  the  contracted  parts,  both  of  the  neck  of  the 


CULTIVATION  WITHOUT  OXYGEN.  187 

tube  and  the  side  arm,  are  then  sealed  in  the  flame.1 
This  tube  can  be  used  for  either  solid  or  liquid  media, 
but,  owing  to  its  usual  small  capacity,  gives  better  re- 
sults with  fluid  media.  (For  precautions  in  using  hydro- 
gen see  note  to  FrankeFs  method,  page  189.) 

Method  of  Buchner.  The  plan  suggested  by  Buchuer 
of  allowing  the  cultures  to  develop  in  an  atmosphere 
robbed  of  its  oxygen  by  pyrogallic  acid  gives  very  good 
results.  In  this  method  the  culture,  which  is  either  a 
slant  or  stab  culture  in  a  test-tube,  is  placed — tube,  cot- 
ton plug,  and  all — into  a  larger  tube  in  the  bottom  of 
which  has  been  deposited  1  gramme  of  pyrogallic  acid 
and  10  c.c.  of  -^  normal2  caustic  potash  solution.  The 
larger  tube  is  then  tightly  plugged  with  a  rubber  stopper. 
The  oxygen  is  quickly  absorbed  by  the  pyrogallic  acid, 
and  the  organisms  develop  in  the  remaining  constituents 
of  the  atmosphere,  viz.,  nitrogen,  a  small  amount  of  CO2, 
and  a  trace  of  ammonia. 

Method  of  C.  Frdnkel.  Carl  Frankel  suggests  the 
following  as  a  modification  of,  or  substitute  for  the 


1  As  the  tubes  come  from  the  maker  the  contracted  parts  marked  x  in  the 
cut  are  usually  so  thick  as  to  render  the  sealing  in  the  flame  during  the  pas- 
sage of  hydrogen  somewhat  troublesome  ;  it  is  better  to  draw  them  out  in  the 
flame  quite  thin  before  passing  the  hydrogen  into  the  tube.    This  makes  the 
final  sealing  a  matter  of  no  difficulty. 

2  A  normal  solution  is  one  that  contains  in  a  litre  as  many  grammes  of  the 
dissolved  substance  as  are  indicated  by  its  molecular  equivalent.  The  equiva- 
lent is  that  amount  of  a  chemical  compound  which  possesses  the  same  chem- 
ical value  as  does  one  atom  of  hydrogen.    For  example :   One  molecule  of 
hydrochloric  acid  (HC1)  has  a  molecular  weight  and  also  an   equivalent 
weight  of  36.5 ;  a  molecule  of  this  acid  has  the  same  chemical  value  as  one  • 
atom  of  hydrogen.    Its  normal  solution  is  therefore  36.5  grammes  to  the  litre. 
On  the  other  hand,  sulphuric  acid  (H2S04)  contains  in  each  molecule  two  re- 
placeable hydrogen  atoms  ;  its  normal  solution  is  not,  therefore,  80  grammes 
(its  molecular  weight)  to  the  litre,  but  that  amount  which  would  be  equiva- 
lent chemically  to  one  hydrogen  atom,  viz.,  40  grammes  (one-half  its  molecu- 
lar weight)  to  the  litre.    A  normal  solution  of  caustic  potash  contains  as 
many  grammes  to  the  litre  as  the  number  of  its  molecular  weight— 56.1 
grammes  to  the  litre  of  water. 


188 


BACTERIOLOGY. 


tubes  of  Liborius :  The  tube  is  first  inoculated  as 
if  it  were  to  be  poured  as  a  plate  or  rolled  as  an  ordi- 
nary Esmarch  tube.  The  cotton  plug  is  then  re- 
placed by  a  rubber  stopper,  through  which  pass  two 
glass  tubes.  These  must  all  have  been  sterilized  in  the 
steam  sterilizer  before  using.  On  the  outer  side  of  the 
stopper  these  two  tubes  are  bent  at  right  angles  to  the 

FIG.  36. 


Frankel's  method  for  the  cultivation  of  anaerobic  bacteria. 

long  axis  of  the  test-tube  into  which  they  are  to  be 
placed,  and  both  are  slightly  drawn  out  in  the  gas 
flame.  At  the  outer  extremity  of  both  of  these  tubes 
a  plug  of  cotton  is  placed ;  this  is  to  prevent  access  of 
foreign  organisms  during  manipulation.  At  the  inner 
side  of  the  rubber  stopper — that  is,  the  end  which  is  to 


CULTIVATION  WITHOUT  OXYGEN.  189 

be  inserted  into  the  test-tube — the  glass  tubes  are  of 
different  lengths :  one  reaches  to  within  0.5  cm.  of  the 
bottom  of  the  test-tube,  the  other  is  cut  off  flush  with 
the  under  surface  of  the  stopper.  The  outer  end  of  the 
longer  glass  tube  is  then  connected  with  a  hydrogen 
generator  and  hydrogen  is  allowed  to  bubble  through 
the  gelatin  (Fig.  36,  A)  in  the  tube  until  all  contained 
air  has  been  expelled  and  its  place  taken  by  the  hydro- 
gen.1 When  the  hydrogen  has  been  bubbling  through 
the  gelatin  for  about  five  minutes  (at  least)  one  can  be" 
reasonably  sure  that  all  oxygen  has  been  expelled.  The 
drawn-out  portions  of  the  tubes  can  then  be  sealed  in  the 
gas-flame  without  fear  of  an  explosion.  The  protruding 
end  of  the  rubber  stopper  is  then  painted  around  with 
melted  paraffin  and  the  tube  rolled  in  the  way  given  for 
ordinary  Esmarch  tubes.  A  tube  thus  prepared  and 
containing  growing  colonies  is  shown  in  Fig.  36,  B. 

The  development  that  now  occurs  is  in  an  atmosphere 
of  hydrogen,  all  oxygen  having  been  expelled.    During 


1  Before  beginning  the  experiment  it  is  always  wise  to  test  the  hydrogen, 
i.  e.,  to  see  that  it  is  free  from  oxygen  and  there  is  no  danger  of  an  explosion, 
for  unless  this  be  done  the  entire  apparatus  may  be  blown  to  pieces  and  a  seri- 
ous accident  occur.  The  agents  used  should  be  pure  zinc,  and  pure  sulphuric 
acid  of  about  25  to  30  per  cent,  strength.  When  the  gas  is  beginning  to  be 
given  off,  the  outlet  of  the  generator  should  be  closed  and  kept  closed  until 
the  gas  reservoir  is  quite  filled  with  hydrogen .  The  outlet  should  then  be 
opened  and  the  entire  volume  of  gas  allowed  to  escape,  care  being  taken  that 
no  flame  is  in  the  neighborhood.  This  should  be  repeated  again,  after  which 
a  sample  of  the  hydrogen  generated  should  be  collected  in  an  inverted  test- 
tube  in  the  ordinary  way  for  collecting  gases  over  water,  viz.,  by  filling  a  test- 
tube  with  water,  closing  its  mouth  with  the  thumb,  inverting  it,  and  placing 
its  mouth  under  water,  when,  after  removing  the  thumb,  the  water  will  be 
kept  in  it  by  atmospheric  pressure.  The  hydrogen  which  is  flowing  from  the 
open  generator  may  be  conveyed  to  the  test-tube  by  a  bit  of  rubber  tubing. 
When  the  water  has  been  replaced  try  the  gas,  by  holding  a  flame  near  the 
open  mouth  of  the  test-tube.  If  no  explosion  occurs,  the  hydrogen  is  safe  to 
use.  Should  there  be  an  explosion  the  generation  of  hydrogen  must  be  con- 
tinued in  the  apparatus  until  it  simply  burns  with  a  colorless  flame  when 
tested  in  a  test-tube. 


190  BACTERIOLOGY, 

the  operation  the  tube  containing  the  liquefied  gelatin 
should  be  kept  in  a  water-bath  at  a  temperature  suffi- 
ciently high  to  prevent  its  solidifying,  and  at  the  same 
time  not  high  enough  to  kill  the  organisms  with  which 
it  has  been  inoculated. 

One  of  the  obstacles  to  the  successful  performance  of 
this  method  is  the  bubbling  of  the  gelatin,  the  foam 
from  which  will  often  fill  the  exit  tube  and  sometimes  be 
forced  from  it.  This  may  be  obviated  by  reversing  the 
order  of  proceeding,  viz. :  Prepare  the  Esmarch  roll 
tube  in  the  ordinary  way  with  the  organisms  to  be 
studied,  using  a  relatively  small  amount  of  gelatin,  so 
as  to  have  as  thin  a  layer  as  possible  when  it  is  rolled. 
Then  replace  the  cotton  plug  with  the  sterilized  rubber 
stopper  carrying  the  glass  tubes  through  which  the 
hydrogen  is  to  be  passed,  and  allow  the  hydrogen  to 
flow  through  just  as  in  the  method  first  given.  The 
gas  now  passes  over  the  gelatin  instead  of  through  it, 
and  consequently  no  bubbling  results.  In  all  other 
respects  the  procedure  is  the  same  as  that  given  by 
Frankel. 

Method  of  Kitasato  and  Weil.  For  favoring  the 
anaerobic  conditions,  Kitasato  and  Weil  have  suggested 
the  addition  to  the  culture  media  of  some  strong  re- 
ducing agent.  They  recommend  formic  acid  in  0.3  to 
0.5  per  cent.;  glucose  in  1.5  to  2  per  cent.,  or  blue 
litmus  tincture  in  5  per  cent,  by  volume.  This  is,  of 
course,  in  addition  to  an  atmosphere  from  which  all 
oxygen  has  been  expelled. 

Esmarch' s  method.  Esmarch's  plan  is  to  prepare  in 
the  usual  way  a  roll  tube  of  the  organisms ;  subject  it 
to  a  low  temperature,  and  while  quite  cold  fill  it  with 
liquefied  gelatin,  which  is  caused  to  solidify t rapidly. 


INDOL  PRODUCTION.  191 

In  this  method  the  colonies  develop  along  the  sides  of 
the  tubes,  and  can  more  easily  be  studied  than  where 
they  are  mixed  through  the  gelatin,  as  in  the  method 
of  Liborius. 

By  some  workers  the  oxygen  is  removed  by  actual 
pumping  with  the  air-pump. 

Many  other  methods  exist  for  this  special  purpose, 
but  for  the  beginner  those  given  will  suffice. 

From  what  has  been  said  it  may  be  inferred  that  the 
cultivation  of  anaerobic  bacteria  is  a  simple  matter  and 
attended  with  but  little  difficulty.  Such  an  inference 
will,  however,  be  quickly  dispelled  when  the  beginner 
attempts  this  part  of  his  work  for  the  first  time,  and 
particularly  when  his  efforts  are  directed  toward  the 
isolation  of  these  forms  from  other  organisms  with 
which  they  are  associated.  The  presence  of  spore- 
forming,  facultative  anaerobes  in  mixed  cultures  is 
always  to  be  suspected,  and  it  is  this  group  that  renders 
the  task  so  difficult.  At  best  the  work  requires  undi- 
vided attention  and  no  small  degree  of  skill  in  bacterio- 
logical technique. 

INDOL  PKODUCTION. — The  production  of  products 
other  than  those  that  give  rise  to  alterations  in  the 
reaction  of  the  media,  and  whose  presence  may  be  de- 
tected by  chemical  reactions,  is  now  a  recognized  step 
in  the  identification  of  different  species  of  bacteria. 
Among  these  chemical  products  there  is  one  that  is 
produced  by  a  number  of  organisms,  and  whose  presence 
may  easily  be  detected  by  its  characteristic  behavior 
when  treated  with  certain  substances.  I  refer  to  the 
body  nitroso-indol,  the  reactions  of  which  were  described 
by  Beyer  in  1869,  and  the  presence  of  which  as  a  pro- 


192  BACTERIOLOGY. 

duct  of  growth  of  certain  bacteria  has  since  furnished  a 
topic  for  coDsiderable  discussion. 

Indol,  the  name  by  which  this  body  is  now  generally 
known,  when  acted  upon  by  reducing  agents,  is  seen  to 
become  of  a  more  or  less  conspicuous  rose  color.  This 
body  was  recognized  as  one  of  the  products  of  growth 
of  the  spirillum  of  Asiatic  cholera  first  by  Poel,  and 
a  short  time  subsequently  by  Bujwid  and  by  Dunham, 
and  for  a  time  was  thought  to  be  peculiarly  character- 
istic of  the  growth  of  this  organism.  It  has  since  been 
found  that  there  are  many  other  bacteria  which  also 
possess  the  property  of  producing  indol  in  the  course  of 
their  development. 

The  method  employed  for  its  detection  is  as  follows  : 
Cultivate  the  organism  for  twenty-four  to  forty-eight 
hours  at  a  temperature  of  37°  C.,  in  the  simple  peptone 
solution  known  as  "  Dunham's  solution  "  (see  formula 
for  this  medium).  This  solution  is  preferred  because 
its  pale  color  does  not  mask  the  rose  color  of  the  reaction 
when  the  amount  of  indol  present  is  very  small. 

Four  tubes  should  always  be  inoculated  and  kept 
under  exactly  the  same  conditions  for  the  same  length 
of  time. 

At  the  end  of  twenty- four  or  forty-eight  hours  the 
test  may  be  made.  Proceed  as  follows  :  To  a  tube  con- 
taining 7  c.c.  of  the  peptone  solution,  but  which  has  not 
been  inoculated,  add  10  drops  of  concentrated  sulphuric 
acid.  To  another  similar  tube  add  1  c.c.  of  a  0.01  per 
cent,  solution  of  sodium  nitrite,  and  afterward  10  drops 
of  concentrated  sulphuric  acid.  Observe  the  tubes  for 
five  to  ten  minutes.  No  alteration  in  their  color  appears, 
or  at  least  there  will  be  no  production  of  a  rose  color. 
They  contain  no  iudol. 


INDOL  PRODUCTION.  193 

Treat  in  the  same  way,  with  the  acid  alone,  two  of 
the  tubes  which  have  been  inoculated.  If  no  rose  color 
appears  after  five  or  ten  minutes,  add  1  c.c.  of  the 
sodium  nitrite  solution.  If  now  no  rose  color  is  pro- 
duced, the  indol  reaction  may  be  considered  as  negative. 
No  indol  is  present. 

If  indol  is  present,  and  the  rose  color  appears  after 
the  addition  of  the  acid  alone,  it  is  plain  that  not  only 
indol  has  been  formed,  but  likewise  a  reducing  body. 
This  is  found,  by  proper  means,  to  be  salts  of  nitrous 
acid.  The  sulphuric  acid  liberates  this  acid  from  its 
salts  and  permits  of  its  reducing  action  being  brought 
into  play. 

If  the  rose  color  appears  only  after  the  addition  of 
both  the  acid  and  the  nitrite  solution,  then  indol  has 
been  formed  during  the  growth  of  the  organisms,  but  no 
nitrites. 

Control  the  results  obtained  by  treating  the  two  re- 
maining cultures  in  the  same  way. 

The  test  is  sometimes  made  by  allowing  concentrated 
acid  to  flow  down  the  sides  and  collect  at  the  bottom  of 
the  tube ;  the  reaction  is  then  seen  as  a  rose-colored 
zone  overlying  the  line  of  contact  of  the  acid  and  cul- 
ture medium.  This  method  is  open  to  the  objection 
that  if  indol  is  present  in  only  a  very  limited  amount, 
the  rose  color  produced  by  it  is  apt  to  be  masked  by  a 
brown  color  that  results  from  the  charring  action  of  the 
concentrated  acid  on  the  other  organic  matters  in  the 
culture  medium,  so  that  its  presence  may  in  this  way 
escape  detection.  In  view  of  this,  Petri  recommends 
the  use  of  dilute  sulphuric  acid.  He  states  that  when 
indol  is  present  the  characteristic  rose  color  appears  a 
little  more  slowly  with  the  dilute  acid,  but  is  more  per- 


1 94  BACTERIOLOG  Y. 

manent,  and  there  is  never  any  danger  of  its  presence 
being  masked  by  the  occurrence  of  other  color  reactions. 
Test  for  Nitrites.  For  this  purpose  Lunkewicz  has 
recently  recommended  the  employment  of  Ilosvay's 
modification  of  the  method  of  Griess.  As  reagents  the 
following  solutions  are  employed  : 

a.  Naphthylamine 0.1  gramme. 

Dist.  water 20.0  c.c. 

Acetic  acid  (25  per  cent,  sol.)        .       .       .    150.0  c.c. 

6.  Sulfanilic  acid         .       .       .       .       .       .       0.5  gramme. 

Acetic  acid  (25  per  cent,  sol.)        .       .       .    150.0  c.c. 

In  preparing  solution  a  the  naphthylamiue  is  dis- 
solved in  20  c.c.  of  boiling  water,  filtered,  allowed  to 
cool,  and  mixed  with  the  dilute  acetic  acid. 

Solutions  a  and  b  are  then  mixed.  The  resulting 
mixture  should  be  colorless.  It  is  best  to  prepare  it 
fresh  as  it  is  needed,  though  if  kept  in  a  closely  stop- 
pered flask  it  retains  its  virtues  for  some  time. 

When  added  to  cultures  containing  nitrites,  in  the 
proportion  of  one  volume  to  five  volumes  of  the  culture, 
a  deep  red  color  appears  in  a  few  seconds.  If  nitrites 
are  not  present  no  color  reaction  occurs.  In  making 
the  test  on  cultures  always  control  the  results  by  tests 
on  the  same  medium  not  inoculated,  as  some  of  the  in- 
gredients of  which  the  medium  is  composed  may  con- 
tain nitrites.  Lunkewicz  recommends  the  use  of  Merck's 
peptone  for  this  test,  claiming  that  nitrites  are  always 
to  be  found  in  Witte's  peptones. 

POINTS  TO  BE  OBSERVED  IN  DESCRIBING  AN  ORGANISM. 

The  following  is  an  outline  of  points  to  be  considered 
in  describing  a  new  organism  or  in  identifying  an  or* 
ganism  with  one  already  described  ; 


DESCRIBING  AN  ORGANISM.  195 

1.  Its  source — as  air,  water,  or  soil.     If  found  in  the 
animal  body,  is  it  normally  present  or  only  in  patholog- 
ical conditions? 

2.  Its  form,  size,  mode  of  development,  occurrence  of 
involution    forms    or   other  variations  in  morphology. 
Grouping,  as  in  pairs,  chains,  clumps,  zoogloea ;  presence 
of  capsule ;    development   and   germination  of  spores  ; 
arrangement  of  flagella. 

3.  Staining  peculiarities — especially  its  reactions  with 
Gram's  (or  Weigert's  fibrin)  stain,  and  peculiar  or  irreg- 
ular modes  of  staining. 

4.  Motility — to  be  determined  on  very  fresh  cultures 
and  on  cultures  in  different  media. 

5.  Its  relation  to  oxygen — is  it  aerobic,  anaerobic,  or 
facultative?    Does  it  develop  in  other  gases,  as  carbonic 
acid,  hydrogen,  etc.  ? 

6.  Both  the  macroscopic  and  microscopic  appearance 
of  its  colonies  on  nutrient  gelatin  and  on  nutrient  agar- 
agar. 

7.  The  appearance  of  its  growth  in  stab  and  slant 
cultures  on  gelatin,  agar-agar,  blood-serum,  and  on  po- 
tato. 

8.  The  character  of  its  growth  in  fluid  media,  as  in 
bouillon,  milk,  litmus  milk,  rosolic-acid-peptone  solu- 
tion, and  in  bouillon  containing  glucose. 

9.  Does  it  grow  best   in    acid,  alkaline,  or   neutral 
media  ? 

10.  Is  the  normal  reaction  of  the  medium  altered  by 
its  growth  ?     Is  its  growth  accompanied  by  the  produc- 
tion of  indol ;  is  the  indol  associated  with  the  coincident 
production  of  nitrites  ? 

11.  Is  its  growth  accompanied  by  the  production  of 
gas,  as  evidenced  by  the  appearance  of  gas-bubbles  in 


196  BACTERIOLOGY. 

the  media — both  in  media  containing  fermentable  sugars 
and  those  from  which  these  bodies  are  absent?  When 
cultivated  in  sugar-bouillon  in  the  fermentation  tube, 
what  production  of  gas  is  evolved  under  known  con- 
ditions ?  How  much  of  this  gas  is  carbonic  acid  and 
how  much  is  explosive? 

12.  At  what  temperature  does  it  thrive  best,  and  the 
lowest  and  highest  temperatures  at  which  it  will  de- 
velop ?  What  is  its  thermal  death-point,  both  by  steam 
and  dry-air  methods  of  determining  this  point? 

Ie3.  What  is  its  behavior  when  exposed  to  chemical 
disinfectants  and  antiseptics  ?  Does  it  withstand  drying 
and  other  injurious  influences,  both  in  the  vegetative  and 
spore  stages  ?  The  germicidal  value  of  the  blood-serum 
of  different  animals  may  also  be  tried  upon  it. 

14.  Its  pathogenic  powers — modes  of  inoculation  by 
which  these  are  demonstrated  ;  quantity  of  material  used 
in  inoculation ;  duration  of  the  disease  and  its  symptoms; 
lesions  produced,  and  distribution  of  the  bacteria  in  the 
inoculated  animal ;  which  animals  are  susceptible  and 
which  immune,  and  the  character  of  its  pathogenic  activ- 
ities?    Variations  in  virulence,  and  the  probable  cause 
to  which  they  are  due.    Can  they  be  produced  artificially 
and  at  will  ? 

15.  The  detection  of  specific,  toxic,  and  immunizing 
products  of  growth. 


CHAPTER    XII. 

Inoculation  of  animals  -Subcutaneous  inoculation ;  intra-venous  injection 
—Inoculation  into  the  great  serous  cavities ;  and  into  the  anterior  chamber  of 
the  eye— Observation  of  animals  after  inoculation. 

AFTER  subjecting  an  organism  to  the  methods  of 
study  that  we  have  thus  far  reviewed,  there  remains  to 
be  tested  its  action  upon  animals — i.  e.,  to  determine  if 
it  possesses  the  property  of  producing  disease  or  not, 
and,  if  so,  what  are  the  pathological  results  of  its 
growth  in  the  tissues  of  these  animals,  and  in  what  way 
must  it  gain  entrance  to  the  tissues  in  order  to  produce 
these  results  ? 

The  mode  of  deciding  these  points  is  by  inoculation, 
which  is  practised  in  different  ways  according  to  cir- 
cumstances. Most  commonly  a  bit  of  the  culture  to  be 
tested  is  simply  introduced  beneath  the  skin  of  the 
animal,  but  in  other  cases  it  may  be  necessary  to  intro- 
duce it  directly  into  the  vascular  or  lymphatic  circula- 
tion or  into  one  or  the  other  of  the  great  serous  cavities ; 
or,  for  still  other  purposes  of  observation,  into  the 
anterior  chamber  of  the  eye,  upon  the  iris. 

SUBCUTANEOUS  INOCULATION  OF  ANIMALS. — The 
animals  usually  employed  in  the  laboratory  for  purposes 
of  inoculation  are  white  mice,  gray  house-mice,  guinea- 
pigs,  rabbits,  and  pigeons. 

For  simple  subcutaneous  inoculation  the  steps  in  the 
process  are  practically  the  same  in  all  cases.  The  hair 
or  feathers  are  to  be  carefully  removed.  If  the  skin 


198  BACTERIOLOGY. 

is  very  dirty  it  may  be  scrubbed  with  soap  and  water. 
Sterilization  of  the  skin  is  impossible,  so  that  it  need 
not  be  attempted.  If  the  inoculation  is  to  be  by  means 
of  a  hypodermic  syringe,  then  a  fold  of  the  skin  may 
be  lifted  up  and  the  needle  inserted  in  the  way  common 
to  this  procedure.  If  a  solid  culture  is  to  be  inoculated, 
a  fold  of  the  skin  may  be  taken  up  with  the  forceps 
and  a  pocket  cut  into  it  with  scissors  which  have  previ- 
ously been  sterilized.  This  pocket  must  be  cut  large 
enough  to  admit  the  end  of  the  needle  without  its 
touching  the  sides  of  the  opening  as  it  is  inserted.  Be- 
neath the  skin  will  be  found  the  superficial  and  deep 
connective-tissue  fascia.  These  must  be  taken  up  with 
sterilized  forceps,  and  with  sterilized  scissors  incised  in 
a  way  corresponding  to  the  opening  in  the  skin.  The 
pocket  is  then  to  be  held  open  with  the  forceps  and  the 
substance  to  be  inserted  is  introduced  as  far  back  under 
the  skin  and  fasciae  as  possible,  care  being  taken  not  to 
touch  the  edges  of  the  wound  if  it  can  be  avoided.  The 
wound  may  then  be  simply  pulled  together  and  allowed 
to  remain.  No  stitching  or  efforts  at  closing  it  are 
necessary,  though  a  drop  of  collodion  over  the  point  of 
operation  may  serve  to  lessen  contamination. 

During  manipulation  the  animal  must  be  held  still. 
For  this  purpose  special  forms  of  holders  have  been 
devised,  but  if  an  assistant  is  to  be  obtained  for  the 
operation,  the  simple  subcutaneous  inoculation  may  be 
made  without  the  aid  of  a  mechanical  holder. 

It  is  at  times,  however,  more  convenient  to  dispense 
with  the  presence  of  an  assistant,  and  several  forms  of 
apparatus  have  been  devised  for  holding  guinea-pigs, 
rats,  rabbits,  etc.  For  small  animals,  such  as  mice  and 
rats,  the  holder  suggested  by  Kitasato  is  very  useful.  It 


SUBC UTANEO US  INOCULATION  OF  ANIMALS.     199 

is  simply  a  metal  plate  attached  to  a  stand  by  a  clamped 
ball-and-socket  joint,  so  that  it  can  be  fixed  in  any  posi- 
tion. It  is  provided  with  a  spring-clip  at  one  end  that 
holds  the  animal  by  the  skin  of  the  neck,  and  at  the 
other  end  with  another  clamp  that  holds  the  tail  of  the 
animal.  This  holder  is  shown  in  Fig.  37. 


FIG.  37. 


Kitasato's  mouse  holder. 

For  larger  animals  the  form  of  holder  shown  in  Fig. 
38  is  commonly  used. 

My  attention  has  recently  been  called  to  a  very  simple 
and  useful  holder  for  guinea-pigs.1  It  consists  of  a  metal 
cylinder  of  about  5  cm.  in  diameter  and  about  13  cm. 
long ;  closed  at  one  end  by  a  perforated  cap  of  either  tin 
or  wire  netting.  Along  the  side  of  this  box  is  a  longi- 

1  So  far  as  I  am  aware  this  apparatus  has  not  been  described.  It  has  recently 
been  brought  to  my  notice  by  Dr.  Lydia  Rabinowitsch,  who  informs  me  that 
it  was  devised  by  Dr.  Voges  and  herself  in  the  Institut  fur  Infectionskrank- 
heiten,  Berlin,  where  it  is  now  in  use.  It  is  with  her  kind  permission  that 
this  description  is  published. 


200  BACTERIOLOGY. 

tudinal  slit  of  12  mm.  wide  that  runs  for  9.5  cm. 
from  within  0.5  mm.  of  the  open  extremity  of  the  cylin- 
der. 

The  animal  is  placed  in  such  a  cylinder  with  its  head 

FIG.  38. 


Holder  for  larger  animals. 

toward  the  perforated  bottom.  It  is  then  easily  possible 
to  make  subcutaneous  inoculation  by  taking  up  a  bit  of 
skin  through  the  slit  in  the  side  of  the  box,  or  to  make 
intra-peritoneal  injection  by  drawing  the  posterior  ex- 
tremities slightly  from  the  box  and  holding  them  steady 
between  the  index  and  second  finger,  as  seen  in  Fig.  39. 
It  is  also  very  convenient  for  use  when  the  rectal  tem- 
perature of  these  small  animals  is  to  be  taken.  The 
manipulations  can  easily  be  made  without  the  aid  of  an 
assistant.  Its  construction  is  best  seen  in  Fig.  39. 

For  ordinary  subcutaneous  inoculations  at  the  root 
of  the  tail  in  mice  a  simple  piece  of  apparatus  consists 
of  a  bit  of  board  of  about  7  x  10  cm.  and  2  cm.  thick, 
upon  which  is  tacked  a  hollow,  tapering  roll  of  wire 
gauze,  a  truncated  cone,  of  about  6  cm.  long  and  of 
about  1.5  cm.  in  diameter  at  one  end  and  2  cm.  at  its 
other  end. 


SUBCUTANEOUS  INOCULATION  OF  ANIMALS.    201 

This  is  tacked  upon  the  board  in  such  a  position  that 
its  long  axis  runs  in  the  long  axis  of  the  board,  being 


FIG. 


The  Voges-Rabinowitsch  holder  for  guinea-pigs. 

equidistant  from  its  two  sides.  Its  small  end  is  placed 
at  the  edge  of  the  board.  The  mouse  is  taken  up  by 
the  tail  by  means  of  a  pair  of  tongs  and  allowed  to 
crawl  into  the  smaller  end  of  this  wire  cone.  When  so 
far  in  that  only  the  root  of  the  tail  projects,  the  animal 
is  then  fixed  in  this  position  by  a  clamp  and  thumb- 


202  BACTERIOLOGY. 

screw,  with  which  the  apparatus  (Fig.  40)  is  provided. 
The  animal  usually  remains  perfectly  quiet  and  may  be 
handled  without  difficulty. 

FIG.  40. 


Mouse-holder,  with  mouse  in  proper  position. 

The  hair  from  over  the  root  of  the  tail  is  to  be  care- 
fully cut  away  with  the  scissors,  and  a  pocket  cut 
through  the  skin  at  this  point.  The  inoculation  is  then 
made  into  the  loose  tissue  under  the  skin  over  this  part 
of  the  back  in  the  way  that  has  just  been  described.  It 
is  best  always  to  insert  the  needle  some  distance  along 
the  spinal  column,  and  thus  deposit  the  material  as  far 
from  the  surface-wound  as  possible. 

As  the  subcutaneous  operation  is  very  simple  and 
takes  only  a  few  moments,  guinea-pigs,  rabbits,  and 
pigeons  may  be  held  by  an  assistant.  The  front  legs 
in  the  one  hand  and  the  hind  legs  in  the  other,  with 
the  animal  stretched  upon  its  back  on  a  table,  is  the 
usual  position  for  the  operation  when  practised  upon 
guinea-pigs  and  rabbits.  The  point  at  which  the  inoc- 
ulations are  commonly  made  is  in  the  abdominal  walls 
either  to  the  right  or  left  of  the  median  line  and  about 
3  cm.  distant.  When  pigeons  are  used  they  are  held 
with  the  legs,  tail,  and.  ends  of  the  wings  in  the  one 
hand,  and  the  head  and  anterior  portion  of  the  body 


INJECTION  INTO  THE  CIRCULATION.          203 

in  the  other,  leaving  the  area  occupied  by  the  pectoral 
muscles,  over  which  the  inoculation  is  to  be  made,  free 
for  manipulation.  The  hair  should  be  closely  cut  with 
the  scissors  in  the  case  of  the  guinea-pigs  and  rabbits, 
and  the  feathers  pulled  out  in  the  case  of  the  pigeon. 

INJECTION  INTO  THE  CIRCULATION. — It  is  not  in- 
frequently desirable  to  inject  the  material  under  consider- 
ation directly  into  the  circulation  of  an  animal.  If  the 
rabbit  is  to  be  employed  for  the  purpose,  the  operation 
is  usually  done  upon  one  of  the  veins  in  the  ear. 

To  those  who  have  had  no  practice  in  this  procedure 
it  offers  a  great  mauy  difficulties ;  but  if  the  directions 
which  will  be  given  are  strictly  observed,  the  greatest 
of  these  obstacles  to  the  successful  performance  of  the 
operations  may  be  overcome. 

When  viewing  the  circulation  in  the  ear  of  the  rabbit 
by  transmitted  light,  three  conspicuous  branches  of  the 
main  vessel  (vena  aurieularis  posterior)  will  be  seen. 
One  runs  about  centrally  in  the  long  axis  of  the  ear, 
one  runs  along  its  anterior  margin,  and  one  along  its 
posterior  margin.  The  central  branch  (ramus  anterior 
of  the  vena  aurieularis  posterior)  is  the  largest  and 
most  conspicuous  vessel  of  the  ear,  and  is,  therefore, 
selected  by  the  inexperienced  as  the  branch  into  which 
it  would  appear  easiest  to  insert  a  hypodermic  needle. 
This,  however,  is  fallacious.  This  vessel  lies  very  loosely 
imbedded  in  connective  tissue,  and  in  efforts  to  intro- 
duce a  needle  into  it,  rolls  about  to  such  an  extent  that 
only  after  a  great  deal  of  difficulty  does  the  experiment 
succeed.  On  the  other  hand,  the  posterior  branch 
(ramus  lateralis  posterior  of  the  vena  aurieularis  poste- 
rior) is  a  very  fine,  delicate  vessel  which  runs  along  the 
posterior  margin  of  the  ear,  and  which  is  so  firmly  fixed 


204  BACTERIOLOGY. 

in  the  dense  tissues  which  surround  it  that  it  is  pre- 
vented from  rolling  about  under  the  point  of  the  needle. 
The  further  away  from  the  mouth  of  the  vessel — that 
is,  the  nearer  we  approach  its  capillary  extremity — the 
more  favorable  become  the  conditions  for  the  success  of 
the  operation. 

Select,  then,  the  very  delicate  vessel  lying  quite  close 
to  the  posterior  margin  of  the  ear,  and  make  the  injec- 
tion as  near  to  the  apex  of  the  ear  as  possible.  The 
injection  is  always  to  be  made  from  the  dorsal  surface 
of  the  ear. 

Of  no  less  importance  than  the  selection  of  the  proper 
vessel  is  the  shape  of  the  point  of  the  needle  employed. 

The  hypodermic  needles  as  they  come  from  the 
makers  are  not  suited  at  all  for  this  operation,  because 
of  the  way  in  which  their  points  are  ground.  If  one 
examines  carefully  the  point  of  a  new  hypodermic 
needle  it  will  be  seen  that  the  long  point,  instead  of 
presenting  a  flat,  slanting  surface,  when  viewed  from 
the  side,  is  more  or  less  of  a  curved  surface.  Now,  in 
efforts  to  introduce  such  a  needle  into  a  vessel  of  very 
small  calibre,  it  is  commonly  seen  that  the  extreme 
point  of  the  needle,  instead  of  remaining  in  the  vessel, 
as  it  would  do  were  it  straight,  very  commonly  projects 
into  the  opposite  wall,  and  as  the  needle  is  inserted 
further  and  further  into  the  tissues,  it  is  usually  pushed 
through  the  vessels  into  the  loose  tissues  beyond,  and 
the  material  to  be  injected  is  deposited  into  these  tissues 
instead  of  into  the  circulation.  If,  on  the  contrary,  the 
slanting  point  of  the  needle  be  ground  down  until  its 
surface  is  perfectly  flat  when  viewed  from  the  side, 
and  no  more  curvature  exists,  then  when  once  inserted 
into  a  vessel  it  usually  remains  there,  and  there  is  no 


INJECTION  INTO  THE  CIRCULATION.          205 

tendency  to  penetrate  through  'the  ^opposite  wall.  We 
never  use  a  new  hypodermic  needle  until  its  point  is 
carefully  ground  down  to  a  perfectly  flat,  slanting  sur- 
face and  no  more  curvature  exists. 

These  differences  may  perhaps  come  out  clearer  if 
represented  diagrammatically. 


FIG.  41. 
a 


Hypodermic  needles  magnified,  a,  Improper  point ;  6,  proper  shape  of  point 

In  Fig.  41,  a,  the  needle  has  the  point  usually  seen 
when  new. 

In  Fig.  41,  6,  the  point  has  been  ground  down  to  the 
shape  best  suited  for  this  operation. 

The  needles  need  not  be  returned  to  the  maker.  One 
can  grind  them  to  the  shape  desired  in  a  few  minutes 
upon  an  oilstone. 

The  size  of  the  needle  is  that  commonly  employed  for 
subcutaneous  injections. 

When  the  operation  is  to  be  performed,  an  assistant 
holds  the  animal  gently  but  firmly  in  the  crouching 
position  upon  a  table.  If  the  animal  does  not  remain 
quiet  it  is  best  to  wrap  it  in  a  towel,  so  that  nothing  but 
its  head  protrudes,  though  in  the  most  cases  we  have 
not  found  this  necessary,  and  particularly  if  the  animal 
has  not  been  excited  prior  to  the  beginning  of  the 
operation. 

The  animal  should  be  placed  so  that  the  ear  upon 
which  the  operation  is  to  be  performed  comes  between 

10 


206  BACTERIOLOGY. 

the  operator  and  the  source  of  light.  This  renders 
visible  by  transmitted  light  not  only  the  coarser  vessels 
of  the  ear,  but  also  their  finer  branches.  The  point  at 
which  the  injection  is  to  be  made  is  to  be  shaved  clean 
of  hair,  by  means  of  a  razor  and  soap. 

The  filled  hypodermic  syringe  is  taken  in  one  hand 
and  with  the  other  hand  the  ear  is  held  firmly.  The 
point  of  the  needle  is  then  inserted  through  the  skin 
and  into  the  finest  part  of  the  ramus  posteriory  the  part 
nearest  the  apex  of  the  ear,  where  the  course  of  the 
vessel  is  nearly  straight.  When  the  point  of  the 
needle  is  in  this  vessel  it  gives  to  the  hand  a  sensation 
quite  different  from  that  felt  when  it  is  in  the  midst  of 
connective  tissue.  As  soon  as  one  thinks  the  point  of 
the  needle  is  in  the  vessel,  a  drop  or  two  of  the  fluid 
may  be  injected  from  the  syringe,  and  if  his  suspicions 
are  correct  the  circulation  in  the  small  ramifications  and 
their  anastomoses  will  quickly  alter  in  appearance. 
Instead  of  their  containing  blood,  the  colorless  fluid 
which  is  being  injected  will  now  be  seen  to  circulate. 
This  must  be  carefully  observed,  for  sometimes  when  the 
needle-point  is  not  actually  in  the  vessel,  but  is  in  the 
lymph-spaces  surrounding  it,  an  appearance  somewhat 
similar  is  to  be  seen.  It  may  always  be  differentiated, 
however,  by  continuing  the  injection,  when  the  circu- 
lation of  clear  fluid  through  the  vessels  will  not  only 
fail  to  take  the  place  of  the  circulating  blood,  but 
there  will  at  the  same  time  appear  a  localized  swelling 
under  the  skin  about  the  point  of  the  needle.  The 
needle  must  then  be  withdrawn  and  inserted  into  the 
vessel  at  a  point  a  little  nearer  to  its  proximal  end. 

Care  must  be  taken  that  no  air  is  injected. 

The  hypodermic  syringe  and  needle  must,  previous 


INJECTION  INTO  THE  CIRCULATION. 


207 


to  operation,  have  been  carefully  sterilized  in  the  steam 
sterilizer  or  in  boiling  water.  The  animal  must  be  kept 
under  close  observation  for  about  an  hour  after  injec- 
tion. 

The  operation  is  one  that  cannot  be  learned  from 
verbal  description.  It  can  only  be  successfully  per- 
formed after  actual  practice. 

If  the  precautions  which  have  been  mentioned  are 
observed,  but  little  difficulty  in  performing  the  opera- 
tion will  be  experienced. 

Its  greater  convenience  and  simplicity  as  compared 
with  other  methods  for  the  introduction  of  substances 
into  the  circulation  commend  it  as  an  operation  with 
which  to  make  oneself  familiar.  The  animals  sustain 
practically  no  wound,  they  experience  no  pain — at  least 
they  give  no  evidence  of  pain — and  no  anaesthesia  is 

required. 

FIG.  42. 


Forms  of  hypodermic  syringe . 
A,  Koch's  syringe  ;  B,  syringe  of  Strohschein ;  C,  Overlack's  lorm. 

The  form  of  syringe  best  suited  for  this  operation  is 
of  the  ordinary  design,  but  one  that  permits  of  thorough 
sterilization  by  steam.  It  should  be  made  of  glass  and 


208  BACTERIOLOGY. 

metal,  with  asbestos  packings.  The  syringes  commonly 
employed  are  those  shown  in  Fig.  42.  — A,  Koch's  ;  B, 
Strohschein's ;  C,  Overlack's. 

For  the  operations  requiring  exact  dosage  experi- 
ence has  led  me  to  prefer  a  syringe  after  the  pattern 
of  0,  in  Fig.  42,  i.  e.y  of  the  design  commonly  used 
by  physicians.  The  reason  for  this  is  as  follows: 
In  making  hypodermic  injections  or  injections  into  the 
circulation,  there  is  a  certain  amount  of  resistance  to  the 
passage  of  fluid  from  the  needle.  If  one  overcome  this 
resistance  by  means  of  a  cushion  of  compressed  air,  as 
is  the  case  in  syringes  A  and  B  of  Fig.  42,  the  sudden 
expansion  of  the  air  in  the  body  of  the  syringe  when 
resistance  is  overcome,  frequently  causes  a  larger  amount 
of  fluid  to  be  ejected  from  the  needle  than  was  desired. 
On  the  other  hand,  no  such  accident  is  likely  to  occur 
when  the  fluid  is  forced  from  the  barrel  of  the  syringe 
by  the  head  of  a  close-fitting  piston,  with  no  air  inter- 
vening between  the  fluid  and  the  head  of  the  piston. 
With  such  an  instrument,  properly  manipulated,  the 
dose  can  always  be  controlled  with  accuracy. 

INOCULATION  INTO  THE  LYMPHATIC  CIRCULATION. 
— Fluid  cultures  or  suspensions  of  bacteria  may  be  in- 
jected into  the  lymphatics  by  way  of  the  testicles.  The 
operation  is  a  simple  one.  One  simply  plunges  the 
point  of  the  hypodermic  needle  directly  into  the  sub- 
stance of  the  testicle  and  then  injects  the  amount 
desired. 

Injections  made  in  this  manner  are  sometimes  fol- 
lowed by  interesting  pathological  lesions  of  the  lym- 
phatic apparatus  of  the  abdomen. 


INOCULATION  INTO  THE  PERITONEUM.       209 
INOCULATION   INTO   THE   GEEAT   SEROUS   CAVITIES. 

Inoculation  into  the  peritoneum  presents  no  difficulties 
if  fluids  are  to  be  introduced.  In  this  case  one  makes, 
with  a  pair  of  hot  scissors,  a  small  nick  through  the 
skin  down  to  the  underlying  fascia,  and,  taking  up  a 
fold  of  the  abdominal  wall  between  the  fingers,  plunges 
the  hypodermic  needle  through  the  opening  just  made 
directly  into  the  peritoneal  cavity.  There  is  no  fear 
of  penetrating  the  intestines  or  other  internal  viscera  if 
the  puncture  be  made  along  the  median  line  at  about 
midway  between  the  end  of  the  sternum  and  the  sym- 
physis  pubis.  Though  this  may  seem  a  rude  method,  it 
is,  nevertheless,  the  rarest  of  accidents  to  find  that  the 
intestines  have  been  penetrated.  The  object  of  the 
primary  incision  is  to  lessen  the  chances  of  contaminat- 
ing the  inoculation  by  bacteria  located  in  the  skin, 
some  of  which  would  adhere  to  the  needle  if  it  were 
plunged  directly  through  the  skin,  and  might  complicate 
the  results. 

If  solid  substances,  bits  of  tissue,  etc.,  are  to  be  intro- 
duced into  the  peritoneum  it  becomes  necessary  to  con- 
duct the  operation  upon  the  lines  of  a  laparotomy.  The 
hair  should  be  shaved  from  a  small  area  over  the  median 
line,  after  which  the  skin  is  to  be  thoroughly  washed. 
A  short  longitudinal  incision  (about  2  cm.  long)  is  then 
to  be  made  in  the  median  line  through  the  skin,  and 
down  to  the  fasciae.  Two  subcutaneous  sutures,  as 
employed  by  Halsted,  are  then  to  be  introduced  trans- 
verse to  the  line  of  incision  at  about  1  cm,  apart,  and 
their  ends  left  loose.  This  particular  sort  of  suture 
does  not  pass  through  the  skin,  but  instead,  the  needle  is 
introduced  into  the  subcutaneous  tissues  along  the  edge 


210 


BACTERIOLOGY. 


of  the  incision.  In  this  case  they  are  to  pass  into  the 
abdominal  cavity  and  out  again,  entering  at  one  side  of 
the  line  of  incision  and  leaving  at  the  other,  as  indicated 
by  the  solid  and  dotted  lines  in  Fig.  43.  (This  figure 
indicates  the  primary  opening  through  the  skin.  By  the 
longitudinal  dotted  line  is  seen  the  opening  to  be  made  into 
the  abdomen  ;  by  the  transverse  dotted  lines,  with  their 
loose  ends,  the  sutures  as  placed  in  position  before  the 
abdomen  is  opened  ;  it  will  be  seen  that  these  sutures  in 
all  cases  pass  through  the  subcutaneous  tissues  only  and 
do  not  penetrate  the  skin  proper.)  The  opening  through 


FIG.  43. 


the  remaining  layers  may  now  be  completed ;  the  bit  of 
tissue  deposited  in  the  peritoneal  cavity,  under  precau- 
tions that  will  exclude  all  else  ;  the  edges  of  the  wound, 
drawn  evenly  and  gently  together  by  tying  the  sutures, 
and  the  lines  of  incision  dressed  with  collodion.  It 
should  be  needless  to  say  that  this  operation  must  be 
conducted  under  the  strictest  precautions,  to  avoid  com- 


ANIMALS  AFTER  INOCULATION.  21 1 

plications.  All  instruments,  sutures,  ligatures,  etc., 
must  be  carefully  sterilized  either  in  the  steam  sterilizer 
for  twenty  minutes,  or  by  boiling  in  2  per  cent,  sodium 
carbonate  solution  for  ten  minutes  ;  the  hands  of  the  oper- 
ator, though  they  should  not  touch  the  wound,  should 
be  carefully  cleansed  and  the  material  to  be  introduced 
into  the  abdomen  should  be  handled  with  only  sterilized 
instruments. 

Inoculation  into  the  pleural  cavity  is  much  less  fre- 
quently called  for — in  fact,  it  is  not  a  routine  method 
employed  in  this  work.  It  is  not  easy  to  enter  the 
pleural  cavity  with  a  hypodermic  needle  without  injur- 
ing the  lung,  and  it  is  rare  that  conditions  call  for  the 
introduction  of  solid  particles  in  this  locality. 

Inoculation  into  the  anterior  chamber  of  the  eye  is  per- 
formed by  making  a  puncture  through  the  cornea  just 
in  front  of  its  junction  with  the  sclerotic,  the  knife  being 
passed  into  the  anterior  chamber  in  a  plane  parallel 
to  the  plane  of  the  iris.  By  the  aid  of  a  fine  pair  of 
forceps  the  bit  of  tissue  is  passed  through  the  opening 
thus  made  and  is  deposited  upon  the  iris,  where  it  is 
allowed  to  remain,  and  where  its  pathogenic  properties 
upon  the  iris  can  be  conveniently  studied.  It  is  a  mode 
of  inoculation  of  very  limited  application,  and  is  there- 
fore but  rarely  practised.  It  was  employed  by  Cohnheim 
in  demonstrating  the  infectious  nature  of  tuberculous 
tissues,  tuberculosis  of  the  iris  being  the  constant  result 
of  the  introduction  of  tuberculous  tissue  into  the 
anterior  chamber  of  the  eye  of  rabbits. 

OBSERVATION   OF   ANIMALS   AFTER    INOCULATION. 

After  either  of  these  methods  of  inoculation,  particu- 
larly when  unknown  species  of  bacteria  are  being  tested, 


212  BACTERIOLOGY. 

the  animal  is  to  be  kept  under  constant  observation  and 
all  that  is  unusual  in  its  conduct  noted — as,  for  instance, 
elevation  of  temperature  ;  loss  of  weight ;  peculiar  posi- 
tion in  its  cage ;  loss  of  appetite ;  roughening  of  the  hair ; 
excessive  secretions,  either  from  the  air-passages,  con- 
junctiva, or  kidneys ;  looseness  of  or  hemorrhage  from 
the  bowels  ;  tumefaction  or  reaction  at  site  of  inoculation, 
etc.  If  death  ensue  in  from  two  to  four  days  it  may 
reasonably  be  expected  that  at  autopsy  evidence  of  either 
acute  septic  or  toxic  processes  will  be  found.  It  some- 
times occurs,  however,  that  inoculation  results  in  the 
production  of  chronic  conditions,  and  the  animal  must 
be  kept  under  observation  often  for  weeks.  In  these 
cases  it  is  important  to  note  the  progress  of  the 
changes  by  their  effect  upon  the  physical  conditions 
of  the  animal,  viz.,  upon  the  nutritive  processes  as 
evidenced  by  fluctuation  in  weight,  and  upon  the  body 
temperature.  For  this  purpose  the  animal  is  to  be 
weighed  daily,  always  at  about  the  same  hour  and 
always  about  midway  between  the  hours  of  feeding ;  at 
the  same  time  its  temperature  as  indicated  by  a  ther- 
mometer placed  in  the  rectum  is  to  be  recorded.1  By 
the  comparison  of  these  daily  observations  with  one  an- 
other, one  is  aided  in  observing  the  course  the  infection 
is  taking. 

Too  much  stress  must  not,  however,  be  laid  upon 
moderate  and  sudden  daily  fluctuations  in  either  tem- 
perature or  weight,  as  it  is  a  common  observation  that 
presumably  normal  animals  when  confined  in  cages  and 

1  The  thermometer  must  be  inserted  into  the  rectum  beyond  the  grasp  of 
the  sphincter,  otherwise  pressure  upon  its  bulb  by  contraction  of  this  muscle 
may  force  up  the  mercurial  column  to  a  point  higher  than  that  resulting 
from  the  actual  body  temperature. 


ANIMALS  AFTER  INOCULATION. 


213 


fed  regularly  often  present  very  striking  temporary  gains 
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214 


BACTERIOLOGY. 


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ANIMALS  AFTER  INOCULATION. 


215 


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BACTERIOLOGY. 


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from  among  the  stock  animals  and  placed  each  in  a  clean 
cage,  the  kind  used  for  animals  under  experiment,  and 


ANIMALS  AFTER  INOCULATION.  217 

kept  under  as  good  general  conditions  as  possible.  For 
the  first  week  the  rabbits  received  each  100  grammes  of 
green  food  (cabbage  and  turnips)  daily,  and  the  guinea- 
pigs  30  grammes  each  of  the  same  food.  During  the 
second  week  this  daily  amount  of  food  was  doubled ; 
during  the  third  week  it  was  quadrupled,  and  for  the 
fourth  and  fifth  weeks  they  each  received  an  excess  of 
food  daily,  consisting  of  green  vegetables  and  grain 
(oats  and  corn).  By  reference  to  the  charts  sudden 
diurnal  fluctuations  in  weight  will  be  observed  that  do 
not  correspond  in  all  instances  with  scarcity  or  sufficiency 
of  food.  With  the  rabbits  there  is  a  gradual  loss  of 
weight  with  the  smaller  amounts  of  food,  which  losses 
are  not  totally  recovered  as  the  food  is  increased.  With 
the  guinea-pigs  there  is  likewise  at  first  a  loss,  but  after 
a  short  time  the  weight  remains  tolerably  constant,  and 
is  not  as  conspicuously  affected  by  the  increase  in  food 
as  one  might  expect.  From  the  recorded  temperatures 
one  sees  the  peculiar  fluctuations  mentioned.  To  just 
what  they  are  due  it  is  impossible  to  say.  It  is  mani- 
fest that  the  normal  temperature  of  these  animals,  if  we 
can  speak  of  a  normal  temperature  for  animals  present- 
ing such  fluctuations,  is  about  a  degree  or  more,  Centi- 
grade, higher  than  that  of  human  beings.  The  animals 
from  which  these  charts  were  made  were  not  inoculated, 
nor  were  they  subjected  to  any  operative  procedures 
whatever,  the  only  deviations  from  normal  conditions 
being  the  variations  in  the  daily  amount  of  food  given. 
In  certain  instances,  however,  there  will  be  noticed  a 
constant  tendency  to  diminution  in  weight,  notwith- 
standing the  daily  fluctuations,  and  after  a  time  a  con- 
dition of  extreme  emaciation  may  be  reached,  the  animal 
often  being  reduced  to  from  50  to  60  per  cent,  of  its 


218  BACTERIOLOGY. 

original  weight.  In  other  cases,  after  inoculations  to 
which  the  animal  is  not  susceptible,  rabbits  in  particular, 
if  properly  fed,  will  frequently  gain  steadily  in  weight. 
The  condition  of  progressive  emaciation  just  mentioned 
is  conspicuously  seen  after  intra-venous  inoculation  of 
rabbits  with  cultures  of  the  bacillus  typhi  abdominalis 
and  of  th  e  bacterium  coli  commune  referred  to  in  the 
chapter  on  the  latter  organism,  and  if  looked  for  will 
doubtless  be  seen  to  follow  inoculation  with  other  organ- 
isms capable  of  producing  chronic  forms  of  infection, 
but  which  are  frequently  considered  non-pathogenic  be- 
cause of  their  inability  to  induce  acute  conditions.  Not 
infrequently  in  chronic  infections  there  may  be  hardly 
any  marked  and  constant  temperature  variations  until 
just  before  death,  when  there  will  sometimes  be  a  rise 
and  at  other  times  a  fall  of  temperature. 

In  the  majority  of  cases,  however,  one  must  be  very 
cautious  as  to  the  amount  of  stress  laid  upon  changes 
in  weight  and  temperature,  for  unless  they  are  progres- 
sive or  continuous  in  one  or  another  direction  they  may 
have  little  or  no  significance  in  indicating  the  existence 
or  absence  of  disease. 


CHAPTER  XIII. 


Post-mortem  examination  of  animals— Bacteriological  examination  of  the 
tissues— Disposal  of  tissues  and  disinfection  of  instruments  after  the  exami- 
nation. 


DURING  the  bacteriological  examination  of  the  tis- 
sues of  dead  animals,  certain  rigid  precautions  must  be 
observed  in  order  to  avoid  error. 

The  autopsy  should  be  made  as  soon  as  possible  after 
death.  If  delay  cannot  be  avoided,  the  animal  should 
be  kept  on  ice  until  the  examination  can  be  made,  other- 
wise decomposition  sets  in,  and  the  saprophytic  bacteria 
now  present  may  interfere  with  the  accuracy  of  results. 

When  the  autopsy  is  to  be  made,  the  animal  is  first 
inspected  externally,  and  all  visible  lesions  noted.  It 
is  then  to  be  fixed  upon  its  back  upon  a  board  with 
nails  or  tacks.  The  four  legs  and  the  end  of  the  nose, 
through  which  the  tacks  are  driven,  are  to  be  moder- 
ately extended.  Plates  are  now  to  be  made  from  the 
site  of  inoculation,  if  this  is  subcutaneous.  The  sur- 
faces of  the  thorax  and  abdomen  are  then  to  be  moist- 
ened to  prevent  the  fine  hairs,  dust,  etc.,  from  floating 
about  in  the  air  and  interfering  with  the  work.  An  in- 
cision is  then  made  through  the  skin  from  the  chin  to 
the  symphysis  pubis.  This  is  only  a  skin  incision,  and 
does  not  reach  deeper  than  the  muscles.  It  is  best  done 
by  first  making  a  small  incision  with  a  scalpel,  just  large 
enough  to  permit  of  the  introduction  of  one  blade  of  a 
blunt-pointed  scissors.  It  is  then  completed  with  the 


220  BACTERIOLOGY. 

scissors.  The  whole  of  the  skin  is  now  to  be  carefully 
dissected  away,  not  only  from  the  abdomen  and  thorax, 
but  from  the  axilliary,  inguinal,  and  cervical  regions,  and 
the  fore  and  hind  legs  as  well.  The  skin  is  then  pinned 
back  to  the  board  so  as  to  keep  it  as  far  from  the  abdo- 
men and  thorax  as  possible,  for  it  is  from  the  skin  that 
the  chances  of  contamination  are  greatest. 

It  now  becomes  necessary  to  proceed  very  carefully. 
All  incisions  from  this  time  on  are  to  made  only 
through  surfaces  that  have  been  sterilized.  The  sterili- 
zation is  best  accomplished  by  the  use  of  a  broad-bladed 
common  knife  that  has  been  heated  in  the  gas-flame. 
The  blade,  made  quite  hot,  is  to  be  held  upon  the 
region  of  the  linea  alba  until  the  skin  at  that  region 
begins  to  burn  ;  it  is  then  held  transverse  to  this  line 
over  about  the  centre  of  the  abdomen,  thus  making  two 
sterilized  tracks  through  which  the  abdomen  may  be 
opened  by  a  crucial  incision.  The  sterilization  thus 
accomplished  is,  of  course,  directed  only  against  organ- 
isms that  may  have  fallen  upon  the  surface  from  with- 
out, and  it  therefore  need  not  extend  deep  down  through 
the  tissues. 

In  the  same  way  two  burned  lines  may  be  made  from 
either  extremity  of  the  transverse  line  up  to  the  top  of 
the  thorax. 

With  a  hot  scissors  the  central  longitudinal  incision, 
extending  from  the  point  of  the  sternum  to  the  geni- 
talia,  is  to  be  made  without  touching  the  internal  vis- 
cera. The  abdominal  wall  must  therefore  be  held  up 
during  the  operation  with  sterilized  forceps  or  hook. 

The  cross  incision  is  made  in  the  same  way.  When 
this  is  completed,  an  incision  through  the  ribs  with  a 


POST-MORTEM  EXAMINATION  OF  ANIMALS.    221 

pair  of  heavy,  sterilized  scissors,  is  made  along  the 
scorched  tracks  on  either  side  of  the  thorax. 

After  this  the  whole  anterior  wall  of  the  thorax  may 
easily  be  lifted  up,  and  by  severing  the  connections  with 
the  diaphragm  it  may  be  completely  removed. 

When  this  is  done  and  the  abdominal  flaps  laid  back, 
the  contents  of  both  cavities  are  to  be  inspected  and 
their  condition  noted  without  disturbing  them. 

After  this,  the  first  steps  to  be  taken  are  to  prepare 
plates  or  Esmarch  tubes  from  the  blood,  liver,  spleen, 
kidneys,  and  any  exudates  that  may  exist. 

This  is  best  done  as  follows : 

Heat  a  scalpel  quite  hot  and  apply  it  to  a  small  sur- 
face of  the  organ  from  which  the  cultures  are  to  be 
made.  Hold  it  upon  the  organ  until  the  surface  directly 
beneath  it  is  visibly  scorched.  Then  remove  it,  heat  it 
again,  and  while  quite  hot  insert  its  point  through  the 
capsule  of  the  organ.  Into  the  opening  thus  made  insert 
a  sterilized  platinum-wire  loop,  made  of  wire  a  little 
heavier  than  that  commonly  employed.  Project  this 
deeply  into  the  tissues  of  the  organ ;  by  twisting  it 
about  enough  material  from  the  centre  of  the  organ  can 
be  obtained  for  making  the  cultures. 

As  the  resistance  offered  by  the  tissues  is  sometimes 
too  great  to  permit  of  a  puncture  with  the  ordinary 
wire  loop,  Nuttall  (Centralblatt  fur  Baderiologie  und 
Parasitenkunde,  1892,  Bd.  xi.  p.  538)  has  devised  for 
the  purpose  a  platinum- wire  spear  which  possesses  con- 
siderable advantage  over  the  loop.  It  is  of  the  form 
seen  in  Fig.  48.  It  is  easily  made  by  beating  a  piece 
of  heavy  platinum  wire  into  a  spear-head  at  one  end, 
and  perforating  this  with  a  small  drill,  as  seen  in  the 


222  BACTERIOLOG  Y. 

cut.  It  is  attached  by  the  other  end  to  either  a  metal 
or  glass  handle,  preferably  the  former.  It  can  be 
readily  thrust  into  the  densest  of  the  soft  tissues,  and 
by  twisting  it  about  after  its  introduction  particles  of 
the  tissue  sufficient  for  examination  are  withdrawn  in 
the  eye  of  the  spear-head. 


FIG.  48. 

C 


Nuttall's  platinum  spear  for  use  at  autopsies. 

The  cultures  from  the  blood  are  usually  made  from 
one  of  the  cavities  of  the  heart,  which  is  always  entered 
through  a  surface  which  has  been  burned  in  the  way 
given. 

In  addition  to  cultures,  cover-slips  from  the  site  of 
inoculation,  from  each  organ  and  from  any  exudates 
that  may  exist,  must  be  made.  These,  however,  are 
prepared  after  the  materials  for  the  cultures  have  been 
obtained. 

They  need  not  be  examined  immediately,  but  may  be 
placed  aside,  under  cover,  on  bits  of  paper  upon  which 
the  name  of  the  organ  from  which  they  were  prepared 
is  written. 

When  the  autopsy  is  complete  and  the  gross  appear- 
ances have  been  carefully  noted,  small  portions  of  each 
organ  are  to  be  preserved  in  95  per  cent,  alcohol  for 
subsequent  examination.  Throughout  the  entire  autopsy 
it  must  be  borne  in  mind  that  all  cultures,  cover-slips, 
and  tissues  must  be  carefully  labelled,  not  only  with  the 
name  of  the  organ  from  which  they  originate,  but  with 


POST-MORTEM  EXAMINATION  OF  ANIMALS.    223 

the  date,  designation  of  the  animal,  etc.,  so  that  an 
account  of  their  condition  after  closer  study  may  be 
subsequently  inserted  in  the  protocol. 

The  cover-slips  are  now  to  be  stained,  mounted,  and 
examined  microscopically,  and  the  results  carefully  noted. 

The  same  may  be  said  for  the  subsequent  study  of  the 
cultures  and  the  hardened  tissues  which  are  to  be  stained 
and  subjected  to  microscopic  examination.  The  results 
of  microscopic  study  of  the  cover-slip  preparations 
and  those  obtained  by  cultures  should  iu  most  cases  cor- 
respond, though  it  not  rarely  occurs  that  bacteria  are 
present  in  such  small  numbers  in  the  tissues  that  their 
presence  may  be  overlooked  microscopically,  and  still 
they  may  appear  in  the  cultures. 

If  the  autopsy  has  been  performed  in  the  proper  way, 
under  the  precautions  given,  and  sufficiently  soon  after 
death,  the  results  of  the  bacteriological  examination 
should  be  either  negative  or  the  organisms  which  appear 
should  be  in  pure  cultures. 

This  is  particularly  the  case  with  the  cultures  made 
from  the  internal  viscera. 

Both  the  cover-slips  and  cultures  made  from  the  point 
of  inoculation  are  apt  to  contain  a  variety  of  organisms. 

If  the  organism  obtained  in  pure  culture  from  the 
internal  viscera,  or  those  predominating  at  the  point  of 
inoculation  of  the  animal,  have  caused  its  death,  then 
subsequent  inoculation  of  pure  cultures  of  this  organ- 
ism into  the  tissues  of  a  second  animal  should  produce 
similar  results. 

When  the  autopsy  is  quite  finished,  the  remainder  of 
the  animal  should  be  burned ;  all  instruments  subjected 
to  either  sterilization  by  steam  or  boiling  for  fifteen 


224  BACTERIOLOGY. 

minutes  in  a  1  to  2  per  cent,  soda  solution,  and  the  board 
upon  which  the  animal  was  tacked,  as  well  as  the  tacks, 
towels,  dishes,  and  all  other  implements  used  at  the 
autopsy,  are  to  be  sterilized  by  steam.  All  cultures, 
cover-slips,  and,  indeed,  all  articles  likely  to  have  infec- 
tious material  upon  them,  must  be  thoroughly  sterilized 
as  soon  as  they  are  of  no  further  service. 


APPLICATION  OF  THE  METHODS  OF 
BACTERIOLOGY. 


CHAPTER  XIV. 

To  obtain  material  with  which  to  begin  work. 

EXPOSE  to  the  air  of  an  inhabited  room  a  slice  of 
freshly  steamed  potato  or  a  bit  of  slightly  moistened 
bread  upon  a  plate  for  about  one  hour.  Then  cover  it 
with  an  ordinary  water-glass  and  place  it  in  a  warm  spot 
(temperature  not  to  exceed  that  of  the  human  body — 
37.5°  C.),  and  allow  it  to  remain  unmolested.  At  the 
end  of  twenty-four  to  thirty-six  hours  there  will  be 
seen  upon  the  cut  surface  of  the  bread  or  potato  small, 
round,  oval,  or  irregularly  round  patches  which  present 
various  appearances. 

These  differences  in  macroscopic  appearance  are  due, 
in  some  cases,  to  the  presence  or  absence  of  color ;  in 
others  to  a  higher  or  lower  degree  of  moisture  ;  in  some 
instances  a  patch  will  be  glistening  and  smooth,  while  its 
neighbor  may  be  dull  and  rough  or  wrinkled  ;  here  will 
appear  an  island  regularly  round  in  outline,  and  there 
an  area  covered  by  an  irregular  ragged  deposit.  All 
of  these  gross  appearances  are  of  value  in  aiding  us  to 
distinguish  between  these  colonies — for  colonies  they 
are — and  under  the  same  conditions  the  organisms  com- 


226  BACTERIOLOGY. 

posing  each  of  them  will  always  produce  growths  of  ex- 
actly the  same  appearauce.  It  was  just  such  an  experi- 
ment as  this,  accidentally  performed,  that  suggested  to 
Koch  a  means  of  separating  and  isolating  from  mixtures 
of  bacteria  the  component  individuals  in  pure  cultures, 
and  it  was  from  this  observation  that  the  methods  of 
cultivation  on  solid  media  were  evolved. 

If,  without  molesting  our  experiment,  we  continue 
the  observation  from  day  to  day,  we  shall  notice  changes 
in  the  colonies  due  to  the  growth  and  multiplication  of 
the  individuals  composing  them.  In  some  cases  the 
colonies  will  always  retain  their  sharply  cut,  round,  or 
oval  outline,  and  will  increase  but  little  in  size  beyond 
that  reached  after  forty-eight  to  seventy-two  hours, 
whereas  others  will  spread  rapidly,  and  will  very  quickly 
overrun  the  surface  upon  which  they  are  growing,  and 
indeed,  grow  over  the  smaller,  less  rapidly  developing 
colonies.  In  a  number  of  instances,  if  the  observation 
be  continued  long  enough,  many  of  these  rapidly  grow- 
ing colonies  will,  after  a  time,  lose  their  lustrous  and 
smooth  or  regular  surface  and  will  show,  at  first  here 
and  there,  elevations  which  will  continue  to  appear  until 
the  whole  surface  takes  on  a  wrinkled  appearance. 
Again  bubbles  may  be  seen  scattered  through  the  colo- 
nies. These  are  due  to  the  escape  of  gas  resulting  from 
fermentation  which  the  organisms  bring  about  in  the 
medium  upon  which  they  are  growing.  Sometimes 
peculiar  odors  resulting  from  the  same  cause  will  be 
noticed. 

Note  carefully  all  these  changes  and  appearances,  as 
they  must  be  employed  subsequently  in  identifying  the 
individual  organisms  from  which  each  colony  on  the 
medium  has  developed. 


MATERIAL  WITH  WHICH  TO  BEGIN  WORK.    227 

If  now  we  examine  these  points  upon  our  bread  or 
potato  with  a  hand-lens  of  low  magnifying  power  we 
will  be  enabled  to  detect  differences  not  noticeable  to  the 
naked  eye.  In  some  cases  we  shall  still  see  nothing  more 
than  a  smooth  non-characteristic  surface;  while  in  others, 
minute,  sometimes  regularly  arranged,  corrugations  may 
be  observed.  In  one  colony  they  may  appear  as  toler- 
ably regular  radii,  radiatiug  from  a  central  spot ;  aud 
again  they  may  appear  as  concentric  rings ;  and  if  by 
the  methods  which  have  been  described  we  obtain  from 
these  colonies  their  individual  components  in  pure  cul- 
ture, we  shall  see  that  this  characteristic  arrangement 
in  folds,  radii,  or  concentric  rings,  or  the  production  of 
color,  is  under  normal  conditions  constant. 

So  much  for  the  simplest  naked-eye  experiment  that 
can  be  made  in  bacteriology,  and  which  serves  to  fur- 
nish the  beginner  with  material  upon  which  to  begin 
his  studies.  It  is  not  necessary  at  this  time  for  him  to 
burden  his  mind  with  names  for  these  organisms ;  it  is 
sufficient  for  him  to  recognize  that  they  are  mostly  of 
different  species  and  that  they  possess  characteristics 
which  will  enable  him  to  differentiate  the  one  from  the 
other. 

In  order  now  for  him  to  proceed  it  is  necessary  that 
he  should  have  familiarized  himself  with  the  methods  by 
which  his  media  are  prepared  and  the  means  employed 
in  sterilizing  them  and  retaining  them  sterile — i.  e.y  of 
preventing  the  access  of  foreign  germs  from  without — 
otherwise  his  efforts  to  obtain  and  retain  his  organisms 
as  pure  cultures  will  be  in  vain. 

EXPOSURE  AND  CONTACT. — Make  a  number  of  plates 
from  bits  of  silk  used  for  sutures,  after  treating  them  as 
follows  : 


228  BACTERIOLOGY. 

Place  some  of  these  pieces  (about  5  centimetres  long) 
into  a  sterilized  test-tube,  and  sterilize  them  by  steam 
for  one  hour.  At  the  end  of  the  sterilization  remove 
one  piece  with  sterilized  forceps  and  allow  it  to  brush 
against  your  clothing,  then  make  a  plate  from  it ;  draw 
another  piece  across  the  table  and  then  plate  it.  Sus- 
pend three  or  four  pieces  upon  a  sterilized  wire  hook 
and  let  them  hang  for  thirty  minutes  free  in  the  air, 
being  sure  that  they  touch  nothing  but  the  hook ;  then 
plate  them  separately. 

Note  the  results. 

In  what  way  do  these  experiments  differ  and  how  can 
the  differences  be  explained  ? 

Expose  to  the  air  six  Petri  dishes  into  which  either 
sterilized  gelatin  or  agar-agar  has  been  poured  and  allowed 
to  solidify ;  allow  them  to  remain  exposed  for  five,  ten, 
fifteen,  twenty,  twenty-five,  and  thirty  minutes,  in  a 
room  where  no  one  is  at  work.  Treat  a  second  set  in 
the  same  way  in  a  room  where  several  persons  are  mov- 
ing about.  Be  careful  that  nothing  touches  them,  and 
that  they  are  exposed  only  to  the  air.  Each  dish  must 
be  carefully  labelled  with  the  time  of  its  exposure. 

Do  they  present  different  results?  What  is  the  reason 
for  this  difference  ? 

Which  predominate,  colonies  resulting  from  the 
growth  of  bacteria,  or  those  from  common  moulds  ? 

How  do  you  account  for  this  condition  ? 


CHAPTER    XV. 

Various  experiments  in  sterilization  by  steam  and  by  hot  air. 

PLACE  in  one  of  the  openings  in  the  cover  of  the  steam 
sterilizer  au  accurate  thermometer ;  when  the  steam  has 
been  streaming  for  a  minute  or  two  the  thermometer  will 
register  100°  C. ;  wrap  in  a  bundle  of  towels  or  rags  or 
pack  tightly  in  cotton  a  maximum  thermometer ;  let  this 
thermometer  be  in  the  centre  of  a  bundle  large  enough 
to  quite  fill  the  chamber  of  the  sterilizer.  At  the  end 
of  a  few  minutes'  exposure  to  the  streaming  steam  re- 
move it ;  it  will  be  found  to  indicate  a  temperature  of 
100°  C. 

Closer  study  of  the  penetration  of  steam  has  taught 
us,  however,  that  the  temperature  which  is  found  at  the 
centre  of  such  a  mass  may  sometimes  be  that  of  the  air 
in  the  meshes  of  the  material,  and  not  that  of  steam, 
and  for  this  reason  the  sterilization  at  that  point  may 
not  be  complete,  because  hot  air  at  100°  C.  has  not  the 
sterilizing  properties  that  steam  at  the  same  temperature 
possesses.  It  is  necessary,  therefore,  that  this  air  should 
be  expelled  from  the  meshes  of  the  material  and  its  place 
taken  by  the  steam  before  sterilization  is  complete.  This 
is  insured  by  allowing  the  steam  to  stream  through  the 
substances  a  few  minutes  before  beginning  to  calculate 
the  time  of  exposure.  There  is  as  yet  no  absolutely 
sure  means  of  saying  that  the  temperature  at  the  centre 
of  the  mass  is  that  of  hot  air  or  of  steam,  so  that  the 

11 


230  BACTERIOLOGY. 

exact  length  of  time  that  is  required  for  the  expulsion 
of  the  air  from  the  meshes  of  the  material  cannot  be 
given. 

Determine  if  the  maximum  thermometer  indicates  a 
temperature  of  100  C.  at  the  centre  of  a  moist  bundle 
in  the  same  way  as  when  a  dry  bundle  was  employed. 

To  about  50  c.c.  of  bouillon  add  about  one  gramme  of 
chopped  hay,  and  allow  it  to  stand  in  a  warm  place  for 
twenty-four  hours.  At  the  end  of  this  time  it  will  be 
found  to  contain  a  great  variety  of  organisms.  Continue 
the  observation,  and  a  pellicle  will  be  seen  to  form  on 
the  surface  of  the  fluid.  This  pellicle  will  be  made  up 
of  rods  which  grow  as  long  threads  in  parallel  strands. 
In  many  of  these  rods  glistening  spores  will  be  seen. 
After  thoroughly  shaking,  filter  the  mass  through  a  fine 
cloth  to  remove  coarser  particles. 

Pour  into  each  of  several  test-tubes  about  10  c.c.  of 
the  filtrate.  Allow  one  tube  to  remain  unmolested  in 
a  warm  place.  Place  another  in  the  steam  sterilizer  for 
five  minutes ;  a  third  for  ten  minutes ;  a  fourth  for 
one-half  hour;  a  fifth  for  one  hour. 

At  the  end  of  each  of  these  exposures  inoculate  a  tube 
of  sterilized  bouillon  from  each  tube.  Likewise  make 
a  set  of  plates  or  Esmarch  tubes  upon  both  gelatin  and 
agar-agar  from  each  tube,  and  note  the  results.  At  the 
same  time  prepare  a  set  of  plates  or  Esmarch  tubes  on 
agar-agar  and  on  gelatin  from  the  tube  which  has  not 
been  exposed  to  the  action  of  the  steam. 

The  plates  or  tubes  from  the  unmolested  tube  will 
present  colonies  of  a  variety  of  organisms ;  separate  and 
study  these. 

Those  from  the  tube  which  has  been  sterilized  for  five 


STERILIZATION  BY  STEAM  AND  BY  HOT  AIE.    231 

minutes  will  present  colonies  in  moderate  numbers,  but, 
as  a  rule,  they  will  represent  but  a  single  organism. 
Study  this  organism  in  pure  cultures. 

The  same  may  be  predicted  for  the  tube  which  has 
been  heated  for  ten  minutes,  though  the  colonies  will  be 
fewer  in  number. 

The  thirty-minute  tube  may  or  may  not  give  one  or 
two  colonies  of  the  same  organism. 

The  tube  which  has  been  heated  for  one  hour  is 
usually  sterile. 

The  bouillon  tubes  from  the  first  and  second  tubes 
which  were  heated  will  usually  show  the  presence  of 
only  one  organism — the  bacillus  which  gave  rise  to  the 
pellicle-formation  in  our  original  mixture.  This  organ- 
ism is  the  bacillus  subtilis,  and  will  serve  as  an  object 
upon  which  to  study  the  difference  in  resistance  toward 
steam  between  the  vegetative  and  spore  stages  of  the 
same  organism. 

Inoculate  about  100  c.c.  of  sterilized  bouillon  with  a 
very  small  quantity  of  a  pure  culture  of  this  organism, 
and  allow  it  to  stand  in  a  warm  place  for  about  six 
hours.  Now  subject  this  culture  to  the  action  of  steam 
for  five  minutes  ;  it  will  be  seen  that  sterilization,  as  a 
rule,  is  complete. 

Treat  in  the  same  way  a  second  flask  of  bouillon,  in- 
oculated in  the  same  way  with  the  same  organism,  but 
after  having  stood  in  a  warm  place  for  from  forty-eight 
to  seventy-two  hours,  that  is,  until  the  spores  have 
formed,  and  it  will  be  found  that  sterilization  is  not 
complete — the  spores  of  this  organism  have  resisted 
the  action  of  steam  for  five  minutes. 

To  determine  if  sterilization  is  complete  always  resort 
to  the  culture  methods,  as  the  macroscopic  and  micro- 


232  BACTERIOLOGY. 

scopic  methods  are  deceptive ;  cloudiness  of  the  media 
or  the  presence  of  organisms  microscopically  does  not 
always  signify  that  the  organisms  possess  the  property 
of  life. 

Inoculate  in  the  same  way  a  third  flask  of  bouillon 
with  a  very  small  drop  from  one  of  the  old  cultures 
upon  which  the  pellicle  has  formed ;  mix  it  well  and 
subject  it  to  the  action  of  steam  for  two  minutes  ;  then 
place  it  to  one  side  for  from  twenty  to  twenty-four 
hours,  and  again  heat  for  two  minutes ;  allow  it  to  stand 
for  another  twenty-four  hours,  and  repeat  the  process  on 
the  third  day.  No  pellicle  will  be  formed,  and  yet  spores 
were  present  in  the  original  mixture,  and,  as  we  have 
seen,  the  spores  of  this  organism  are  not  killed  by  an 
exposure  of  five  minutes  to  the  steam.  How  can  this 
result  be  accounted  for  ? 

Saturate  several  pieces  of  cotton  thread,  each  about 
2  cm.  long,  in  the  original  decomposed  bouillon,  and  dry 
them  carefully  at  the  ordinary  temperature  of  the  room, 
then  at  a  little  higher  temperature — about  40°  C. — to 
complete  the  process.  Regulate  the  temperature  of  the 
hot-air  sterilizer  for  about  100°  C.,  and  subject  several 
pieces  of  this  infected  and  dried  thread  to  this  tempera- 
ture for  the  same  lengths  of  time  that  we  exposed  the 
same  organisms  in  bouillon  to  the  steam,  viz. :  five,  ten, 
thirty,  and  sixty  minutes.  At  the  end  of  each  of  these 
periods  remove  a  bit  of  thread,  and  prepare  a  set  of 
plates  or  Esmarch  tubes  from  it.  Are  the  results  anal- 
ogous to  those  obtained  when  steam  was  employed  ? 

Increase  the  temperature  of  the  dry  sterilizer  and 
repeat  the  process.  Determine  the  temperature  and 
time  necessary  for  the  destruction  of  these  organisms 


STERILIZATION  BY  STEAM  AND  BY  HOT  AIR.    233 

by  the  dry  heat.  These  threads  should  not  be  simply 
laid  upon  the  bottom  of  the  sterilizer,  but  should  be  sus- 
pended from  a  glass  rod,  which  may  be  placed  inside 
the  oven,  extending  across  its  top  from  one  side  to  the 
other. 

Place  several  of  the  infected  threads  in  the  centre  of 
a  bundle  of  rags.  Subject  this  to  a  temperature  neces- 
sary to  sterilize  the  threads  by  the  dry  method.  Treat 
another  similar  bundle  to  sterilization  by  steam.  In 
what  way  do  the  results  of  the  two  processes  differ  ? 


CHAPTER   XVI. 


Suppuration— The  staphylococcus  pyogeues  aureus— Staphylococcus  pyo- 
genes  albus  and  citreus — Streptococcus  pyogenes — Bacillus  pyocyanus — Gen- 
eral remarks. 


PREPARE  from  the  pus  of  an  acute  abscess  or  boil, 
that  has  been  opened  under  antiseptic  precautions,  a  set 
of  plates  of  agar-agar.  Care  must  be  taken  that  none 
of  the  antiseptic  fluid  gains  access  to  the  culture  tubes, 
otherwise  its  antiseptic  effect  may  be  seen  and  the  de- 
velopment of  the  organisms  interfered  with.  It  is  best, 
therefore,  to  take  up  a  drop  of  the  pus  upon  the  plati- 
num-wire loop  after  it  has  been  flowing  for  a  few 
seconds;  even  then  it  must  be  taken  from  the  mouth 
of  the  wound  and  before  it  has  run  over  the  surface  of 
the  skin.  At  the  same  time  prepare  two  or  three  cover- 
slips  from  the  pus. 

Microscopic  examination  of  these  slips  will  reveal  the 
presence  of  a  large  number  of  pus-cells,  both  multi-nu- 
cleated and  with  horseshoe- shaped  nuclei,  some  threads 
of  disintegrated  and  necrotic  connective  tissue,  and  ly- 
ing here  and  there  throughout  the  preparation,  small 
round  bodies  which  will  sometimes  appear  singly,  some- 
times in  pairs,  and  frequently  will  be  seen  grouped  to- 
gether somewhat  like  clusters  of  grapes.  (See  Fig.  49.) 
They  stain  readily  and  are  commonly  located  in  the  ma- 
terial between  the  pus-cells;  very  rarely  they  may  be 
seen  in  the  protoplasmic  body  of  the  cell.  (Compare  the 
preparation  with  a  similar  one  made  from  the  pus  of 


SUPPURATION.  235 

gonorrhoea — see  Fig.  50.      In  what  way  do  the  two 
preparations  differ,  the  one  from  the  other  ?) 


FIG.  49. 

C 


Preparation  from  pus,  showing  pus-cells,  A,  and  staphylococci,  C. 

After  twenty-four  hours  in  the  incubator  the  plates 
will  be  seen  to  be  studded,  here  and  there,  with  yellow  or 


PIG.  50. 


f  I    |    _ 


if       tf%ji 

v  "•••/ 


Pus  of  gonorrhoea,  showing  diplococci  in  the  bodies  of  the  pus-cells. 

orange-colored  colonies,  which  are  usually  round,  moist, 
and  glistened  in  their  naked-eye  appearances.     When 


236  BACTERIOLOGY. 

located  in  the  depths  of  the  medium  they  are  commonly 
seen  to  be  lozenge  or  whetstone  in  shape,  while  often 
they  appear  as  irregular  stars  with  blunt  points,  and 
again  as  irregularly  lobulated  dense  masses.  In  struc- 
ture they  are  conspicuous  for  their  density.  Under  the 
low  objective  they  appear,  when  on  the  surface,  as 
coarsely  granular,  irregularly  round  patches,  with  more 
or  less  ragged  borders  and  a  dark  irregular  central 
mass,  which  has  somewhat  the  appearance  of  masses  of 
coarser  clumps  of  the  same  material  as  that  composing 
the  rest  of  the  colony.  Microscopically,  these  colonies 
are  composed  of  small  round  cells,  irregularly  grouped 
together.  They  are  in  every  way  of  the  same  appear- 
ance as  those  seen  upon  the  original  cover-slip  prepara- 
tions. 

Prepare  from  one  of  these  colonies  a  pure  stab  culture 
in  gelatin.  After  thirty-six  to  forty-eight  hours  lique- 
faction of  the  gelatin  along  the  track  of  the  needle, 
most  conspicuous  at  its  upper  end,  will  be  observed.  As 
growth  continues  the  liquefaction  becomes  more  or  less 
of  a  stocking-shape,  and  gradually  widens  out  at  its  up- 
per end  into  an  irregular  funnel.  This  will  continue 
until  the  whole  of  the  gelatin  in  the  tube  eventually 
becomes  fluid.  There  can  always  be  noticed  at  the 
bottom  of  the  liquefying  portion  an  orange-colored  or 
yellow  mass  composed  of  a  number  of  the  organisms 
which  have  sunk  to  the  bottom  of  the  fluid. 

On  potato  the  growth  is  quite  luxuriant,  appearing 
as  a  brilliant,  orange-colored  layer,  somewhat  lobulated 
and  a  little  less  moist  than  when  growing  upon  agar- 
agar.  It  does  not  produce  fermentation  with  gas- 
production.  It  belongs  to  the  group  of  facultative 
aerobes. 


STAPHYLOCOCCUS  PYOGENES  AUREUS.      237 

In  milk  it  rapidly  brings  about  coagulation  with  acid 
reaction. 

It  is  not  motile,  and  being  of  the  family  of  micro- 
cocci,  does  not  form  endogenous  spores.  It  possesses, 
however,  a  marked  resistance  toward  detrimental  agen- 
cies. 

In  bouillon  it  causes  a  diffuse  clouding,  and  after  a 
time  presents  a  yellow  sedimentation. 

This  organism  is  the  commonest  of  the  pathogenic 
bacteria  with  which  we  shall  meet.  It  is  the  staphylo- 
COCGUS  pyogenes  aureus,  and  is  the  organism  most  fre- 
quently concerned  in  the  production  of  acute,  circum- 
scribed, suppurative  inflammations.  It  is  almost  every- 
where present,  and  is  the  organism  that  causes  the  sur- 
geon so  much  annoyance. 

In  studying  its  effects  upon  lower  animals  a  number 
of  points  are  to  be  remembered.  While  it  is  the  etio- 
logical  factor  in  the  production  of  most  of  the  suppura- 
tive processes  in  man,  still  it  is  with  no  little  difficulty 
that  these  conditions  can  be  reproduced  in  lower  animals. 
Its  subcutaneous  introduction  into  their  tissues  does  not 
always  result  in  abscess-formation,  and  when  it  does, 
there  seems  to  have  been  some  coincident  interference 
with  the  circulation  and  nutrition  of  these  tissues  which 
renders  them  less  able  to  resist  its  inroads.  When  in- 
troduced into  the  great  serous  cavities  of  the  lower 
animals  its  presence  here,  too,  is  not  always  followed  by 
the  production  of  inflammation.  If  the  abdominal  cavity 
of  a  dog,  for  example,  be  carefully  opened  so  as  to  make  as 
slight  a  wound  as  possible,  and  no  injury  be  done  to  the 
intestines,  large  quantities  of  bouillon  cultures  or  watery 
suspensions  of  this  organism  may,  and  repeatedly  have 
been  introduced  into  the  peritoneum  without  the  slight- 

11* 


238  BACTERIOLOGY. 

est  injury  to  the  animal.  On  the  contrary,  if  some 
substance  which  acts  as  a  direct  irritant  to  the  intes- 
tines— such,  for  example,  as  a  small  bit  of  potato  upon 
which  the  organisms  are  growing — be  at  the  same  time 
introduced,  or  the  intestines  be  mechanically  injured, 
so  that  there  is  a  disturbance  in  their  circulation,  then 
the  introduction  of  these  organisms  is  promptly  followed 
by  acute  and  fatal  peritonitis.  (Halsted.1) 

On  the  other  hand,  the  results  which  follow  their 
introduction  into  the  circulation  are  practically  constant. 
If  one  inject  into  the  circulation  of  the  rabbit  through 
one  of  the  veins  of  the  ear,  or  in  any  other  way,  from 
0.1  to  0.3  c.c.  of  a  bouillon  culture  or  watery  suspension 
of  a  virulent  variety  of  this  organism,  a  fatal  pyaemia 
always  follows  in  from  two  and  one-half  to  three  days. 
A  few  hours  before  death  the  animal  is  frequently  seen 
to  have  severe  convulsions.  Now  and  then  excessive 
secretion  of  urine  is  noticed.  The  animal  may  appear 
in  moderately  good  condition  until  from  eight  to  ten 
hours  before  death.  At  the  autopsy  a  typical  picture 
presents ;  the  voluntary  muscles  are  seen  to  be  marked 
here  and  there  by  yellow  spots,  which  average  the  size 
of  a  flaxseed,  and  are  of  about  the  same  shape.  They 
lie  usually  with  their  long  axis  running  longitudinally 
between  the  muscle  fibres.  As  the  abdominal  and  thor- 
acic cavities  are  opened  the  diaphragm  is  often  seen  to 
be  studded  by  them.  Frequently  the  pericardial  sac  is 
distended  with  a  clear  gelatinous  fluid,  and  almost  con- 
stantly the  yellow  points  are  to  be  seen  in  the  myocar- 
dium. The  kidneys  are  rarely  without  them  ;  here  they 


1  Halsted  :  The  Johns  Hopkins  Hospital  Reports.    Report  in  Surgery  No.  1, 
1891,  Vol.  II.,  No.  5,  pp.  301-303. 


STUDY  OF  COVER-SLIPS  AND  SECTIONS.      239 

appear  on  the  surface,  scattered  about  as  single  yellow 
points,  or  again,  are  seen  as  conglomerate  masses  of 
small  yellow  points  which  occupy,  as  a  rule,  the  area 
fed  by  a  single  vessel.  If  one  make  a  section  into  one 
of  these  yellow  points  it  will  be  seen  to  extend  deep 
down  through  the  substance  of  the  kidney  as  a  yellow, 
wedge-shaped  mass,  the  base  of  the  wedge  being  at  the 
surface  of  the  organ. 

It  is  very  rare  that  these  abscesses — for  abscesses  the 
yellow  points  are,  as  we  shall  see  when  we  come  to 
study  them  more  closely — are  found  either  in  the  liver, 
spleen,  or  brain ;  their  usual  location  being,  as  said,  in 
the  kidney,  myocardium,  and  voluntary  muscles. 

These  minute  abscesses  contain  a  dry,  cheesy,  necrotic 
centre,  in  which  the  staphylococci  are  present  in  large 
numbers,  as  may  be  seen  upon  cover-slips  prepared  from 
them.  They  may  also  be  obtained  in  pure  culture  from 
these  suppurating  foci. 

Preserve  in  Miiller's  fluid  and  in  alcohol  duplicate 
bits  of  all  the  tissue  in  which  the  abscesses  are  located. 

When  these  tissues  are  hard  enough  to  cut,  sections 
should  be  made  through  the  abscess-points,  and  the  his- 
tological  changes  carefully  studied. 

MICROSCOPIC  STUDY  OF  COVER- SLIPS  AND  SECTIONS. 
— In  cover-slip  preparations  this  organism  stains  readily 
with  the  ordinary  dyes. 

In  tissues,  however,  it  is  best  to  employ  some  method 
by  means  of  which  contrast  stains  may  be  employed, 
and  the  location  and  grouping  of  the  organisms  in  the 
tissues  rendered  more  conspicuous. 

When  stained,  sections  of  tissues  containing  these 
small  abscesses  present  the  following  appearances : 

To  the  naked  eye  will  be  seen  here  and  there  in  the 


240  BACTERIOLOGY. 

section,  if  the  abscesses  are  very  numerous,  small,  darkly 
stained  areas  which  range  in  size  from  that  of  a  pin- 
point up  to  those  having  a  diameter  of  from  1  to  2  mm. 
These  points,  when  in  the  kidney,  may  be  round  or  oval 
in  outline,  or  may  appear  wedge-shaped,  with  the  base 
of  the  wedge  toward  the  surface  of  the  organ.  The 
differences  in  shape  depend  frequently  upon  the  direc- 
tion in  which  the  section  has  been  made  through  the 
kidney.  In  the  muscles  they  are  irregularly  round  or 
oval. 

When  quite  small  they  appear  to  the  naked  eye  as 
simple,  round  or  oval,  darkly  stained  points,  but  when 
they  are  more  advanced  a  pale  centre  can  usually  be 
made  out. 

When  magnified,  they  appear  in  the  earliest  stages 
as  minute  aggregations  of  small  cells,  the  nuclei  of 
which  stain  intensely.  Almost  always  there  can  be 
seen  about  the  centre  of  these  cell-accumulations  evi- 
dences of  progressing  necrosis.  The  normal  structure 
of  the  cells  of  the  tissue  will  be  more  or  less  destroyed ; 
there  will  be  seen  a  granular  condition  due  to  cell-frag- 
mentation ;  at  different  points  about  the  centre  of  this 
area  the  tissue  will  appear  cloudy  and  the  tissue-cells 
will  not  stain  readily.  All  about  and  through  this  spot 
will  be  seen  the  nuclei  of  pus-cells,  many  of  which  are 
undergoing  disintegration.  In  the  smallest  of  these 
beginning  abscesses  the  staphylococci  are  to  be  seen 
scattered  about  the  centre  of  the  necrotic  tissue,  but  in 
a  more  advanced  stage  they  are  commonly  seen  massed 
together  in  very  large  numbers  in  the  form  commonly 
referred  to  as  emboli  of  micrococd. 

The  localized  necrosis  of  the  tissues  which  is  seen  at 
the  centre  of  the  abscess  is  the  direct  result  of  the 


STUDY  OF  COVER-SLIPS  AND  SECTIONS.      241 

action  of  a  poison  produced  by  the  bacteria,  and  repre- 
sents the  starting-point  for  all  abscess-formations. 

When  the  process  is  somewhat  advanced  the  different 
parts  of  the  abscess  are  more  easily  detected.  They  then 
present  in  sections  somewhat  the  following  conditions : 
At  the  centre  can  be  seen  a  dense,  granular  mass,  which 
stains  readily  with  the  basic  aniline  dyes  and,  when 
highly  magnified,  is  found  to  be  made  up  of  staphy- 
lococci.  Sometimes  the  shape  of  this  mass  of  staphylo- 
cocci  corresponds  to  that  of  the  capillary  in  which  the 
organisms  became  lodged  and  developed.  Immediately 
about  the  embolus  of  cocci  the  tissues  are  seen  to  be  in 
an  advanced  stage  of  necrosis.  Their  structure  is 
almost  completely  destroyed,  though  it  is  seen  to  be 
more  advanced  in  some  of  the  elements  of  the  tissues 
than  in  others.  As  we  approach  the  periphery  of  this 
faintly  stained  necrotic  area,  it  becomes  marked  here 
and  there  with  granular  bodies,  irregular  in  size  and 
shape,  which  stain  in  the  same  way  as  do  the  nuclei  of 
the  pus-cells  and  represent  the  result  of  disintegration 
going  on  in  these  cells. 

Beyond  this  we  come  upon  a  dense,  deeply  stained 
zone,  consisting  of  closely  packed  pus-cells;  of  granular 
detritus  resulting  from  destructive  processes  acting  upon 
these  cells ;  and  of  the  normal  cellular  and  connective 
tissue  elements  of  the  part.  Here  and  there  through 
this  zone  will  be  seen  localized  areas  of  beginning  death 
of  the  tissues.  This  zone  gradually  fades  away  into  the 
healthy  surrounding  tissues.  It  constitutes  the  so-called 
"  abscess-wall." 

Such  is  the  picture  presented  by  the  miliary  abscess 
when  produced  experimentally  in  the  rabbit,  and  it 
corresponds  throughout  with  the  pathological  changes 


242  BACTERIOLOGY. 

which  accompany  the  formation  of  larger  abscesses  in 
the  tissues  of  human  beings. 

From  these  small  abscesses  in  the  tissues  of  the  rabbit 
the  staphylococcus  pyogenes  aureus  may  again  be  ob- 
tained in  pure  culture,  and  will  present  identically  the 
same  characteristics  that  were  possessed  by  the  culture 
with  which  the  animal  was  inoculated. 

THE  LESS  COMMON  PYOGENIC  ORGANISMS. — The 
pus  of  an  acute  abscess  in  the  human  being  may  some- 
times contain  other  organisms  beside  the  staphylococcus 
pyogenes  aureus.  The  staphylococcus  pyogenes  albus 
and  citreus  may  be  found.  The  colonies  of  the  former 
are  white,  those  of  the  latter  are  lemon-color.  With 
these  exceptions  they  are  in  all  essential  cultural  pecu- 
liarities similar  to  the  staphylococcus  aureus.  As  a  rule 
they  are  not  virulent  for  animals,  and  when  they  do 
possess  pathogenic  properties,  it  is  in  a  much  lower  de- 
gree than  is  commonly  the  case  with  the  golden  staphy- 
lococcus. The  streptococcus  pyogenes  is  also  sometimes 
present.  The  commonest  of  the  pyogenic  organisms, 
however,  is  that  just  described,  viz. :  the  staphylococcus 
pyogenes  aureus. 

THE  STREPTOCOCCUS  PYOGENES. — From  a  spread- 
ing phlegmonous  inflammation  prepare  cover-slips  and 
cultures.  What  is  the  predominating  organism  ?  Does 
it  appear  in  the  form  of  irregular  clusters  like  those  of 
grapes,  or  have  its  individuals  a  definite  regular  arrange- 
ment? Are  its  colonies  like  those  of  the  staphylococcus 
pyogenes  aureus  f 

Isolate  this  organism  in  pure  cultures.  In  these  cul- 
tures it  will  be  found  on  microsopic  examination  to 
present  an  arrangement  somewhat  like  a  chain  of  beads. 
(Fig.  51.) 


THE  STREPTOCOCCUS  PYOGENES.  243 

Determine  its  peculiarities  and  describe  them  accu- 
rately. They  should  be  as  follows  : 

Upon  microscopic  examination  a  micrococcus  should 
be  found,  but  differing  in  its  arrangement  from  the 
staphylococci  just  described.  The  single  cells  are  not 
scattered  irregulary  or  arranged  in  clumps  similar  to 
bunches  of  grapes,  but  "are  joined  together  in  chains  like 
strands  of  beads.  These  strands  are  sometimes  regular 
in  the  arrangement  and  size  of  the  individual  cells  com- 
posing them,  but  more  commonly  certain  irregular  parts 
may  be  seen  in  them.  Here  they  appear  as  if  two  or 

FIG.  51. 


Streptococcus  pyogenes. 

three  cells  had  fused  together  to  form  a  link,  so  to 
speak,  in  the  chain,  that  is  somewhat  longer  than  the 
remaining  links ;  again,  portions  of  the  chain  may  be 
thinner  than  the  rest,  or  may  appear  broken  or  ragged. 
Commonly  the  individuals  comprising  this  chain  of  cocci 
are  not  round,  but  appear  flattened  on  the  sides  adjacent 
to  one  another.  The  chains  are  sometimes  short,  con- 
sisting of  four  to  six  cells,  or  again  they  may  be  much 
longer,  and  extend  from  a  half  to  two-thirds  of  the  way 
across  the  field  of  the  microscope. 

Under  artificial  conditions  it  sometimes  grows  well, 
and  can  be  cultivated  through  many  generations,  while 
again  it  rapidly  loses  its  vitality.  When  isolated  from 


244  BACTERIOLOGY. 

the  diseased  area  upon  artificial  media  it  seems  to  retain 
its  vitality  for  a  longer  period  if  replanted  upon  fresh 
media  every  day  or  two  for  a  time ;  but  if  the  first 
generation  is  not  treated  in  this  way,  but  allowed  to  re- 
main upon  the  original  medium,  it  is  not  uncommon  to 
find  the  organism  incapable  of  further  cultivation  after 
a  week  or  ten  days. 

Under  no  conditions  is  the  growth  of  this  organism 
very  luxuriant. 

On  gelatin  plates  its  colonies  appear  after  forty-eight 
to  seventy-two  hours  as  very  small,  flat,  round  points,  of 
a  bluish-white  or  opalescent  appearance.  They  do  not 
cause  liquefaction  of  the  gelatin,  and  in  size  they  rarely 
exceed  0.6—0.8  mm.  in  diameter.  Under-low  magnify- 
ing power  they  have  a  brownish  or  yellowish  tinge  by 
transmitted  light,  and  are  finely  granular.  As  the 
colonies  become  older  their  regular  border  may  become 
slightly  irregular  or  notched. 

In  stab  cultures  in  gelatin  they  grow  along  the  entire 
needle-track  as  a  finely  granular  line,  the  granules  rep- 
resenting minute  colonies  of  the  organism.  On  the 
surface  the  growth  does  not  usually  extend  beyond  the 
point  of  puncture. 

On  agar-agar  plates  the  colonies  appear  as  minute 
pearly  points,  which  when  slightly  magnified  are  seen 
to  be  finely  granular,  of  a  light-brownish  color,  and 
regular  at  their  margins. 

When  smeared  upon  the  surface  of  agar-agar  or  gela- 
tin slants  the  growth  that  results  is  a  thin,  pearly,  finely 
granular  layer,  consisting  of  minute  colonies  growing 
closely  side  by  side.  Its  growth  is  most  luxuriant  on 
glycerin  agar-agar  at  the  temperature  of  the  incubator 
(37.  5°  C.),  and  least  on  gelatin. 


THE  STREP10COCCUS  PYOGENES.  245 

On  blood-serum  its  colonies  present  little  that  is  char- 
acteristic ;  they  appear  as  small,  moist,  whitish  points, 
from  0.6  to  0.8  mm.  in  diameter,  that  are  slightly  ele- 
vated above  the  surface  of  the  serum.  They  do  not 
coalesce  to  form  a  layer  over  the  surface,  but  remain  as 
isolated  colonies. 

On  potato  no  visible  development  appears,  but  after 
a  short  time  (thirty-six  to  seventy-two  hours)  there  is  a 
slight  increase  of  moisture  about  the  point  inoculated, 
and  microscopic  examination  shows  that  a  multiplication 
of  the  organisms  placed  at  this  point  has  occurred. 

In  milk  its  conduct  is  not  always  the  same,  some 
cultures  causing  a  separation  of  the  milk  into  a  firm  clot 
and  colorless  whey,  while  others  do  not  produce  this 
coagulation.  The  latter,  when  cultivated  in  milk  of  a 
neutral  or  slightly  alkaline  reaction,  to  which  a  few 
drops  of  litmus  tincture  have  been  added,  produce  a  very 
faint  pink  color  after  twenty-four  hours  at  37.5°  C. ; 
there  is  no  coagulation. 

In  bouillon  it  grows  as  tangled  masses  or  clumps, 
which  upon  microscopic  examination  are  seen  to  consist 
of  long  chains  of  cocci  twisted  or  matted  together. 

It  grows  best  at  the  temperature  of  the  body  (37.5° 
C.),  and  develops,  but  less  rapidly,  at  the  ordinary  room 
temperature.  When  virulent,  its  virulence  is  said  by 
Petruschky  to  be  preserved  by  retaining  cultures  in  the 
ice-chest  after  they  have  been  growing  on  gelatin  for  two 
days  at  22°  C. 

It  is  a  facultative  anaerobe. 

It  stains  with  the  ordinary  aniline  dyes,  and  is  not 
decolorized  when  subjected  to  Gram's  method. 

It  is  not  motile,  and,  being  a  micrococcus,  does  not 
form  endogeneous  spores.  Under  artificial  conditions  we 


246  BACTERIOLOGY. 

have  no  reason  to  believe  that  it  enters  a  stage  where  its 
resistance  to  detrimental  agencies  is  increased.  In  the 
tissues  of  the  body,  however,  it  appears  to  possess  a 
marked  tenacity  to  vitality,  for  it  is  not  rare  to  observe 
recurrences  of  inflammatory  conditions  due  to  this  organ- 
ism, often  at  a  relatively  long  time  after  the  primary 
site  of  infection  is  healed. 

When  introduced  into  the  tissues  of  lower  animals  its 
effects  are  uncertain.  Rosenbach  and  Passet  claimed 
that  protracted,  progressive,  erysipelatoid  inflammations 
were  produced,  and  Fehleisen,  who  described  a  strepto- 
coccus in  erysipelas  that  is  in  all  probability  identical 
with  the  streptococcus  pyogenes  now  under  considera- 
tion, stated  that  it  produced  in  the  tissues  of  rabbits 
(the  base  of  the  ear)  a  sharply  defined,  migratory  red- 
dening without  pus-formation.  As  a  rule,  it  is  difficult 
to  obtain  auy  definite  pathological  alterations  in  the  tis- 
sues of  animals  through  the  introduction  into  them  of 
cultures  of  this  organism  by  any  of  the  methods  of  in- 
oculation ordinarily  practised. 

This  is  the  streptococcus  pyogenes,  and  is  the  organism 
most  commonly  found  in  rapidly  spreading  suppuration 
in  contradistinction  to  the  staphylococcus  pyogenes 
aureus,  which  is  most  frequently  found  in  circumscribed 
abscess-formations  :  they  may  be  found  together. 

If  the  opportunity  presents,  obtain  cultures  from  a 
case  of  erysipelas.  Compare  the  organism  thus  obtained 
with  the  streptococcus  just  mentioned.  Inoculate  rab- 
bits both  subcutaneously  and  into  the  circulation  with 
about  0.2  c.c.  of  pure  cultures  of  these  organisms  in 
bouillon.  Do  the  results  correspond,  and  do  they  in 
any  way  suggest  the  results  obtained  with  the  staphylo- 
coccus pyogenes  aureus  when  introduced  into  animals 


THE  STREPTOCOCCUS  PJOGENES.  247 

in  the  same  way  ?  Do  these  streptococci  flourish  readily 
on  ordinary  media? 

The  organisms  that  have  just  been  described  are  com- 
monly known  as  the  "  pyogenic  cocci "  of  Ogston, 
Rosen  bach,  and  Passet,  and  up  to  as  late  as  1885  were 
believed  to  be  the  specific  factors  concerned  in  the  pro- 
duction of  suppurative  inflammations.  Since  that  time, 
however,  considerable  modification  of  this  view  has 
taken  place,  and  while  they  are  still  known  to  be  the 
most  common  causes  of  suppuration,  they  are  also  known 
not  to  be  the  only  causes  of  this  process. 

With  the  more  general  application  of  bacteriological 
methods  to  the  study  of  the  manifold  conditions  coming 
under  the  eye  of  the  physician,  the  surgeon,  and  the 
pathologist,  observations  are  constantly  being  made  that 
do  not  accord  with  the  view  formerly  held  with  regard 
to  the  specific  relation  of  the  pyogeuic  cocci  to  all  forms 
of  suppuration.  There  is  an  abundance  of  evidence 
now  at  command  to  justify  the  opinion  that  there  are  a 
number  of  organisms  not  commonly  classed  as  pyogenic 
which  may,  under  peculiar  circumstances,  assume  this 
property.  For  example : 

The  bacillus  of  typhoid  fever  has  been  found  in  pure 
culture  in  osteomyelitis  of  the  ribs ;  in  acute  purulent 
otitis  media ;  in  abscess  of  the  soft  parts ;  in  the  pus  of 
empyema,  and  in  localized  fibro-peritonitis,  either  during 
its  course  or  as  a  sequela  of  typhoid  fever. 

The  bacterium  coli  commune  has  been  found  to  be 
present  in  pure  culture  in  acute  peritonitis;  in  liver 
abscess ;  in  purulent  inflammation  of  the  gall-bladder 
and  ducts  ;  in  appendicitis ;  and  Welch1  has  found  it  in 

1  Welch:    "Conditions  Underlying  the  Infection  of  Wounds,"  American 
Journal  of  the  Medical  Sciences,  November,  1891. 


248  BACTERIOLOGY. 

pure  culture  in  fifteen  different  inflammatory  condi- 
tions. 

The  micrococcus  lanceolatus  (pneumococcus)  has  been 
found  to  be  the  only  organism  present  in  abscess  of  the 
soft  parts  ;  in  purulent  infiltration  of  the  tissues  about 
a  fracture;  in  purulent  cerebro-spinal  meningitis;  in 
suppurative  synovitis ;  in  acute  pericarditis,  and  in  acute 
inflammation  of  the  middle  ear. 

Moreover,  many  of  the  less  common  organisms  have 
been  detected  in  pure  cultures  in  inflammatory  con- 
ditions with  which  they  were  not  previously  thought  to 
be  concerned,  and  to  which  they  are  not  usually  related 
etiologically. 

In  consideration  of  such  evidence  as  this,  it  is  plain 
that  we  can  no  longer  adhere  rigidly  to  the  opinions 
formerly  held  upon  the  etiology  of  suppuration,  but 
must  subject  them  to  modifications  in  conformity  with 
this  newer  evidence.  We  now  know  that  there  exist 
bacteria  other  than  the  "  pyogenic  cocci/7  which,  though 
not  normally  pyogenic,  may  give  rise  to  tissue  changes 
indistinguishable  from  those  produced  by  the  ordinary 
pus  organisms.1 

THE  BACILLUS  PYOCYANUS  (BACILLUS  OF  GREEN  PUS). 

Another  common  organism  that  may  properly  be 
mentioned  at  this  place,  though  perhaps  not  strictly 
pyogeuic,  is  a  bacillus  frequently  found  in  discharges 
from  wounds,  viz.,  the  bacillus  pyocyanus,  or  bacillus  of 
green  pus,  or  of  blue  pus,  or  of  blue-green  pus,  as  it  is 

1  For  a  more  detailed  discussion  of  the  subject,  see  "  The  Factors  Concerned 
in  the  Production  of  Suppuration,"  International  Med.  Mag.,  Phlla.,  May, 
1892. 


THE  BA  CILL  US  PYOCYA  NUS.  249 

commonly  called.  The  bacillus  pyocyanus  is  a  delicate 
rod  with  rounded  or  pointed  ends.  It  is  actively  motile ; 
does  not  form  spores.  As  seen  in  preparations  made 
from  cultures  it  is  commonly  clustered  together  in 
irregular  masses.  It  does  not  form  long  filaments, 
there  being  rarely  more  than  four  joined  together  end  to 
end,  and  most  frequently  not  even  two. 

It  grows  readily  on  all  artificial  media,  and  gives  to 
some  of  them  a  bright-green  color  that  is  most  con- 
spicuous where  it  is  in  contact  with  the  air.  This  green 
color  is  not  seen  in  the  growth  itself  to  any  extent,  but 
is  diffused  through  the  medium  on  which  the  organism 
is  developing.  With  time  this  color  becomes  much 
darker,  and  in  very  old  agar-agar  cultures  may  become 
almost  black  (sometimes  very  dark  blue-green,  at  others 
brownish-black). 

Its  growth  on  gelatin  in  stab  cultures  is  accompanied 
by  liquefaction,  and  the  diffusion  of  a  bright-green  color 
throughout  the  unliquefied  medium.  As  liquefaction 
continues,  and  the  entire  gelatin  ultimately  becomes 
fluid,  the  green  color  is  confined  to  the  superficial  layers 
that  are  in  contact  with  the  air.  The  form  taken  by  the 
liquefying  portion  of  the  gelatin  in  the  earliest  stages  of 
development  is  somewhat  that  of  an  irregular,  slender 
funnel.  (See  Fig.  52.) 

On  gelatin  plates  the  colonies  develop  rapidly ;  they 
are  not  sharply  circumscribed,  but  usually  present  at 
first  a  fringe  of  delicate  filaments  about  their  periphery 
(see  Fig.  53).  As  growth  progresses  and  liquefaction 
becomes  more  advanced,  the  central  mass  of  the  colony 
sinks  into  the  liquefied  depression,  while  at  the  same 
time  there  is  an  extension  of  the  colony  laterally.  At 
this  stage  the  colony,  when  slightly  magnified,  may  pre- 


250 


BACTERIOLOGY. 


sent  various  appearances,  the  most  common  being  that 
shown  in  Fig.  54. 

The  gelatin  between  the  growing  colonies  takes  on  a 
bright  yellowish-green  color,  but  as  growth  is  compar- 
atively rapid,  it  is  quickly  entirely  liquefied,  and  one 
often  sees  the  colonies  floating  about  in  the  pale-green 
fluid. 


FIG.  52. 


FIG.  53. 


Colony  of  b.  pyocyanus  after  twenty-four 
hours  on  gelatin  at  20°-22°  C. 


FIG.  54. 


Stab  culture  of  b. 
pyocyanus  in  gel- 
atin after  twenty- 
eight  hours  at  22°  C. 


Colony  of  b.  pyocyanus  after  forty-two  hours 
on  gelatin  at  20°-22°  C. 


On  agar-agar  the  growth  is  dry,  sometimes  with  a 
slight  metallic  lustre,  and  is  of  a  whitish  or  greenish- 
white  color,  while  the  surrounding  agar-agar  is  bright 
green.  With  time  this  bright  green  becomes  darker, 
passing  to  blue-green,  and  finally  turns  almost  black. 


THE  BACILLUS  PYOOYANUS.  251 

On  potato  the  growth  is  brownish,  dry,  and  slightly 
elevated  above  the  surface.  With  some  cultures  the 
potato  about  the  growth  becomes  green  ;  with  others  this 
change  is  not  so  noticeable.  With  many  cultures  a 
peculiar  phenomenon  may  be  produced  by  lightly  touch- 
ing the  growth  with  a  sterilized  platinum  needle.  This 
phenomenon  consists  in  a  change  of  color  from  brown 
to  green  at  the  point  touched.  It  is  best  seen  in  cultures 
that  have  been  kept  in  the  incubator  for  from  seventy- 
two  to  ninety-six  hours.  It  occurs  in  from  one  to  three 
minutes  after  touching  with  the  needle,  and  may  last 
from  ten  minutes  to  half  an  hour.  This  is  the  "  cha- 
meleon phenomenon  "  of  Paul  Ernst. 

In  bouillon  the  green  color  appears,  and  the  growth 
is  seen  in  the  form  of  delicate  flocculi.  A  very  delicate 
mycoderma  is  also  produced. 

In  milk  it  causes  an  acid  reaction,  with  coincident 
coagulation  of  the  casein. 

On  blood  serum  and  egg  albumin  its  growth  is  accom- 
panied by  liquefaction.  The  growth  on  coagulated  egg 
albumin  is  seen  as  a  dirty-gray  deposit  surrounded  by  a 
narrow  brownish  zone ;  the  remaining  portion  of  the 
medium  is  bright  green  in  color.  As  the  culture  becomes 
older  the  green  may  give  way  to  a  brown  discoloration. 

In  peptone  solution  (double  strength)  it  causes  a 
bluish-green  color.  In  one  of  four  cultures  from  dif- 
ferent sources  there  was  a  blue  color  produced. 

It  produces  indol. 

It  stains  with  the  ordinary  dyes,  and  its  flagella  may 
be  readily  demonstrated  by  Lreffler's  method  of  staining. 

Inoculation  into  animals.  As  a  rule,  cultures  of  this 
organism  obtained  directly  from  the  discharges  of  a 
wound  are  capable,  when  introduced  into  animals,  of 


252  BACTERIOLOGY. 

lighting  up  diseased  conditions ;  but  cultures  that  are 
kept  on  artificial  media  for  a  long  time  may  in  part,  or 
completely,  lose  this  power. 

When  guinea-pigs  or  rabbits  are  inoculated  subcuta- 
neously  with  1  c.c.  of  virulent  fluid  cultures  of  this 
organism,  death  usually  results  in  from  eighteen  to 
thirty-six  hours.  At  the  seat  of  inoculation  there  is 
found  an  extensive  purulent  infiltration  of  the  tissues 
and  a  marked  zone  of  inflammatory  oedema. 

When  introduced  directly  into  the  peritoneal  cavity 
the  results  are  also  fatal,  and  at  autopsy  a  genuine 
fibrinous  peritonitis  is  found.  There  is  usually  an 
accumulation  of  serum  in  both  the  peritoneal  and  pleural 
cavities.  At  autopsies  after  both  methods  of  inoculation 
the  organisms  will  be  found  in  the  blood  and  internal 
viscera  in  pure  cultures. 

When  animals  are  inoculated  with  small  doses  (less 
than  1  c.c.  of  a  bouillon  culture)  of  this  organism, 
death  may  not  ensue,  and  only  a  local  inflammatory 
reaction  (abscess  formation)  may  be  set  up.  In  these 
cases  the  animals  are  usually  protected  against  subse- 
quent inoculation  with  doses  that  would  otherwise  prove 
fatal. 

Most  interesting  in  connection  with  the  bacillus  pyo- 
cyanus  is  the  fact,  as  brought  out  in  the  experiments  of 
Bouchard,  and  of  Charrin  and  others,  that  its  products 
possess  the  power  of  counteracting  the  pathogenic  activ- 
ities of  the  bacillus  anthracis.  That  is  to  say,  if  an 
animal  be  inoculated  with  a  virulent  anthrax  culture, 
and  soon  after  be  inoculated  with  a  culture  of  the  bacillus 
pyocyanus,  the  fatal  effects  of  the  former  inoculation 
may  be  prevented. 

In  the  literature  upon  the  green-producing  organisms 


THE  BA  CILL  US  PYOCYANUS.  253 

that  have  been  found  in  inflammatory  conditions,  sev- 
eral varieties — believed  to  be  distinct  species — have  been 
described,  but  when  cultivated  side  by  side  their  bio- 
logical differences  are  seen  to  be  so  slight  as  to  render  it 
probable  that  they  are  but  modifications  of  one  and  the 
same  species. 


12 


CHAPTER   XVII. 

Sputum  septicaemia— Septicaemia  resulting  from  the  presence  of  the  micro- 
coccus  tetragenus  in  the  tissues— Tuberculosis. 

OBTAIN  from  a  tuberculous  patient  a  sample  of  fresh 
sputum — that  of  the  morning  is  preferable.  Spread 
it  out  in  a  thin  layer  upon  a  black  glass  plate  and 
select  one  of  the  small,  white,  cheesy  masses  or  dense 
mucous  clumps  that  will  be  seen  scattered  through  it. 
With  a  pointed  forceps  smear  it  carefully  upon  two 
or  three  thin  cover-slips,  dry  and  fix  them  in  the 
way  given  for  ordinary  cover-slip  preparations.  Stain 
one  in  the  ordinary  way  with  Loeffler's  alkaline  methy- 
lene-blue  solution,  the  other  by  the  Gram  method,  the 
third  after  the  method  given  for  tubercle  bacilli  in  fluids 
or  sputum. 

In  that  stained  by  Loeffler's  method — slip  No.  1 — 
will  be  seen  a  great  variety  of  organisms — round  cells, 
ovals,  short  and  long  rods,  perhaps  spiral  forms.  But 
not  infrequently  will  be  seen  diplococci,  having  more 
or  less  of  a  lancet  shape ;  they  will  be  joined  together 
by  their  broad  ends,  the  points  of  the  lancet  being  away 
from  the  point  of  juncture  of  the  two  cells.  There  may 
also  be  seen  masses  of  cocci  which  are  conspicuous  for 
their  arrangement  into  groups  of  fours,  the  adjacent 
surfaces  being  somewhat  flattened.  They  are  not  sar- 
cina,  as  one  can  see  by  the  absence  of  the  division  in 
the  third  direction  of  space — they  divide  only  in  two 
directions. 


SPUTUM  SEPTICAEMIA.  255 

In  the  slip  stained  by  the  Gram  method  the  same 
groups  of  the  cocci  which  grow  as  threes  and  fours  will 
be  seen,  but  our  lancet-shaped  diplococci  will  now  pre- 
sent an  altered  appearance — there  can  now  be  detected 
a  capsule  surrounding  them.  This  capsule  is  very 
delicate  in  structure,  and  though  a  frequent  accompani- 
ment is  not  constant.  It  can  sometimes  be  demonstrated 
by  the  ordinary  methods  of  staining,  though  the  method 
of  Gram  is  most  satisfactory.  (Fig.  56.) 

In  the  third  slip  which  has  been  stained  by  the 
method  given  for  tubercle  bacilli  in  sputum,  if  de- 
colorization  has  been  properly  conducted  and  no  con- 
trast stain  has  been  employed,  the  field  will  be  color- 
less or  of  only  a  very  pale  rose  color.  None  of  the 
numerous  organisms  seen  in  the  first  slip  can  now  be 
detected,  but  instead  there  will  be  seen  scattered  through 
the  field  very  delicate  stained  rods,  which  present,  in 
most  instances,  a  conspicuous  beaded  arrangement  of 
their  protoplasm — that  is,  the  staining  is  not  homoge- 
neous, but  at  tolerably  regular  intervals  along  each  rod 
there  are  seen  alternating  intervals  of  light  and  color. 
These  rods  may  be  found  singly,  in  groups  of  twos  or 
threes,  or  sometimes  in  clumps  consisting  of  large  num- 
bers. When  in  two  or  threes  it  is  not  uncommon  to 
find  them  describing  an  X  or  a  V  in  their  mode  of  ar- 
rangement, or  again  they  will  be  seen  lying  parallel  the 
one  to  the  other. 

If  contrast  stains  are  used,  these  rods  will  be  detected 
and  recognized  by  their  retaining  the  original  color  with 
which  they  have  been  stained,  whereas  all  other  bac- 
teria in  the  preparation,  as  well  as  the  tissue-cells  which 
are  in  the  sputum,  will  take  up  the  contrast  color. 
(Fig.  55.) 


256  BACTERIOLOGY. 

These  delicate  beaded  rods  are  the  bacillus  tubercu- 
losis. The  lancet-shaped  diplococci  with  the  capsule 
are  the  micrococcus  lanceolatus. 

FIG.  55. 


Tuberculous  sputum  stained  by  Gabbett's  method.    Tubercle  bacilli  seen  as 
red  rods  ;  all  else  is  stained  blue. 

The  cocci  grouped  in  fours  are  the  micrococcus  tetra- 
genus. 

INOCULATION  EXPERIMENT.  —  Inoculate  into  the 
subcutaneous  tissues  of  a  guinea-pig  one  of  the  small 
white  caseous  masses  similar  to  that  which  has  been 
examined  microscopically.  If  death  ensues  it  will  be 
the  result  of  one  of  the  three  following  forms  of 
infection  : 

a.  Septicaemia1  resulting  from  the  introduction  into 
the  tissues,  of  an  organism  frequently  present  in  the 
sputum.  It  exists  under  the  various  names  :  micro- 
coccus  of  sputum  septicaemia  ;  diplococcus  pneumonia  ; 
pneumococcus  of  Frankel  ;  meningococcus  ;  strepto- 
coccus lauceolatus  Pasteuri  ;  micrococcus  lanceolatus  ; 

1  Septicaemia  is  that  form  of  infection  in  which  the  blood  is  the  chief  field 
of  activity  of  the  organisms. 


SPUTUM  SEPTICAEMIA.  257 

micrococcus  Pasteuri ;  coccus  lanceolatus  ;  bacillus  sali- 
varius  septicus ;  bacillus  septicus  sputigenous ;  diplo- 
coccus  lanceolatus  capsulatus;  micrococcus  pneumonia 
crouposaB. 

6.  A  form  of  septica3mia  resulting  from  the  invasion 
of  the  tissues  by  an  organism  frequently  seen  in  the 
sputum  of  tuberculous  subjects.  It  is  characterized  by 
its  tendency  to  divide  into  fours.  It  is  the  micrococcus 
tetragenus. 

c.  Local  or  general  tuberculosis. 

a.  SPUTUM  SEPTICAEMIA. 

If  at  the  end  of  twenty-four  to  thirty-six  hours  the 
animal  be  found  dead,  we  may  safely  suspect  that  the 
result  was  produced  by  the  introduction  into  the  tissues 
of  the  organism  of  sputum  septicaBmia  above  mentioned, 
viz.,  the  micrococcus  lanceolatus,  which  is  not  uncom- 
monly found  in  the  mouth  of  healthy  individuals  as 
well  as  in  other  conditions. 

Inspection  of  the  seat  of  inoculation  usually  reveals 
a  local  reaction.  "  This  may  be  of  a  serous,  fibrinous, 
hemorrhagic,  necrotic,  or  purulent  character.  Fre- 
quently we  may  find  combinations  of  these  conditions, 
such  as  fibro-purulent,  fibrino-serous,  or  sero-hemor- 
rhagic." l  The  most  conspicuous  naked-eye  change 
undergone  by  the  internal  organs  will  be  enlargement 
of  the  spleen.  It  is  usually  swollen,  but  may  at  times 
be  normal  in  appearance.  It  is  sometimes  hard,  dark 
red,  and  dry,  or  it  may  be  soft  and  rich  in  blood.  Fre- 
quently there  is  a  limited  fibrinous  exudation  over  por- 
tions of  the  peritoneum. 

1  Welch  :  Johns  Hopkins  Hospital  Bulletin,  December,  1892,  vol.  iii.  No.  27. 


258  BACTERIOLOGY. 

Except  in  the  exudations,  the  organisms  are  found 
only  in  the  lumen  of  the  blood  vessels,  where  they  are 
usually  present  in  enormous  numbers. 

In  the  blood  they  are  practically  always  free  and  are 
but  rarely  found  within  the  bodies  of  leucocytes. 

In  stained  preparations  from  the  blood  and  exudates 
a  capsule  is  not  infrequently  seen  surrounding  the  organ- 
isms. (Fig.  56.)  This,  however,  is  not  constant. 

FIG.  56. 


Micrococcus  lanceolatus  in  blood  of  rabbit.    Stained  by  method  of  Gram. 
Decolorization  not  complete. 

If  a  drop  of  blood  from  this  animal  be  introduced 
into  the  tissues  of  a  second  animal  (mouse  or  rabbit) 
identically  the  same  conditions  will  be  reproduced. 

If  the  organism  be  isolated  from  the  blood  of  the 
animal  in  pure  culture,  and  a  portion  of  this  culture  be 
introduced  into  the  tissues  of  a  susceptible  animal,  again 
we  shall  see  the  same  pathological  picture. 

It  must  be  remembered,  however,  that  this  organism 
when  cultivated  for  a  time  on  artificial  media  rapidly 
loses  its  pathogenic  properties.  If,  therefore,  failure  to 
reproduce  the  disease  after  inoculation  from  old  cultures 


SPUTUM  SEPTIC^HIA.  259 

should  occur,  it  is  in  all  probability  due  to  a  disappear- 
ance of  virulence  from  the  organism. 

This  organism  was  discovered  by  Sternberg  in  1880. 
It  was  subsequently  described  by  A.  Frankel  as  the 
etiological  factor  in  the  production  of  acute  fibrinous 
pneumonia. 

It  is  not  uncommonly  present  in  the  saliva  of  healthy 
individuals,  having  been  found  by  Sternberg  in  the  oral 
cavity  of  about  20  per  cent,  of  healthy  persons  examined 
by  him.  It  is  constantly  to  be  detected  in  the  rusty 
sputum  of  patients  suffering  from  acute  fibrinous  pneu- 
monia. Its  presence  has  been  detected  in  the  middle 
ear,  in  the  pericardial  sac,  in  the  pleura,  in  the  serous 
cavities  of  the  brain,  and  indeed  it  may  penetrate  from 
its  primary  seat  in  the  mouth  to  almost  any  of  the  more 
distant  organs. 

The  organism  is  commonly  found  as  a  diplococcus, 
though  here  and  there  short  chains  of  four  to  six  indi- 
viduals joined  together  may  be  detected.  (Fig.  56, 
page  258.)  The  morphology  of  the  individual  cells  is 
more  or  less  oval,  or,  more  strictly  speaking,  lancet- 
shaped,  for  at  one  end  it  is  commonly  pointed.  When 
joined  in  pairs  the  junction  is  always  between  the  broad 
ends  of  the  ovals,  never  between  the  pointed  extremi- 
ties. 

As  already  stated,  in  preparations  directly  from  the 
sputum  or  from  the  blood  of  animals,  a  delicate  capsule 
may  frequently  be  seen  surrounding  them.  Though 
fairly  constant  in  preparations  directly  from  the  blood 
of  animals  and  from  the  sputum  or  lungs  of  pneumonic 
patients,  the  capsule  is  but  rarely  observed  in  artificial 
cultures.  Occasionally  in  cultures  on  blood-serum,  in 
milk,  and  on  agar-agar  they  can,  according  to  some 


260  BACTERIOLOGY. 

authors,  be  detected ;  but  this  is  by  no  means  constant 
or  frequent. 

This  organism  grows  under  artificial  conditions  very 
slowly,  and  frequently  not  at  all. 

When  successfully  grown  upon  the  different  media  it 
presents  somewhat  the  following  appearance  : 

On  gelatin  it  grows  very  slowly,  if  at  all,  probably 
owing  in  part  to  the  low  temperature  at  which  gelatin 
cultures  must  be  kept.  If  development  occurs  it  appears 
as  minute  whitish  or  blue-white  points  on  the  plates. 
These  very  small  colonies  are  round,  finely  granular, 
sharply  circumscribed,  and  slightly  elevated  above  the 
surface  of  the  gelatin.  The  growth  is  very  slow  and  no 
liquefaction  of  the  gelatin  accompanies  it. 

If  grown  in  slant  or  stab  cultures  the  surface  devel- 
opment is  very  limited  ;  along  the  needle-track  tiny 
whitish  or  bluish-white  granules  appear. 

On  nutrient  agar-agar  the  colonies  are  almost  trans- 
parent ;  they  are  more  or  less  glistening  and  very  deli- 
cate in  structure.  On  blood-serum  development  is  more 
marked,  though  still  extremely  feeble.  Here  it  also  ap- 
pears as  a  cluster  of  isolated  fine  points  growing  closely 
side  by  side. 

A  growth  on  potato  is  not  usually  observed.  When 
grown  in  milk  it  commonly  causes  an  acid  reaction  with 
coincident  coagulation  of  the  casein.  Some  varieties, 
especially  non-virulent  ones,  do  not  coagulate  milk.1  It 
is  not  motile. 

It  grows  best  at  a  temperature  of  from  35°  to  38°  C. 
Under  24°  C.  there  is  usually  no  development,  but  in 
a  few  cases  it  has  been  seen  to  grow  at  as  low  a  tem- 

i  Welch,  loc.  cit. 


SPUTUM  SEPTIC^MIA.  261 

perature  as  18°  C.     From  42°  C.  on,  the  development 
is  checked. 

Under  most  favorable  conditions  the  growth  is  very 
slow.  It  grows  as  well  without  as  with  oxygen.  It  is, 
therefore,  one  of  the  facultative  anaerobic  forms. 

The  most  successful  eiforts  at  the  cultivation  of  this 
organism  are  those  seen  when  the  agar-agar-gelatin 
mixture  of  Guarniari  is  employed.  (See  this  medium.) 

It  may  be  stained  with  the  ordinary  aniline  staining 
reagents.  For  demonstration  of  the  capsule  the  method 
of  Gram  gives  the  best  results.  (See  Stainings.) 

This  organism  is  conspicuous  for  the  irregularity  of 
its  behavior  when  grown  under  artificial  conditions; 
usually  it  loses  its  pathogenic  properties  after  a  few 
generations ;  but  again  this  peculiarity  may  be  retained 
for  a  much  longer  time.  Not  rarely  it  fails  to  grow  after 
three  or  four  transplantations  on  artificial  media,  though 
at  times  it  may  be  carried  through  many  generations. 

Inoculation  into  animals.  The  results  of  inoculations 
with  pure  cultures  of  this  organism  are  also  conspicuous 
for  their  irregularity.  Most  commonly  when  the  organ- 
ism is  of  full  virulence  the  form  of  septicaemia  just  de- 
scribed is  produced,  but  at  times  it  is  found  to  be  totally 
devoid  of  pathogenic  powers ;  between  these  extremes 
cultures  may  be  obtained  possessing  all  variations  in  the 
intensity  of  their  disease-producing  properties.  The 
principal  pathological  conditions  that  may  be  produced 
by  this  organism  by  inoculations  into  animals,  accord- 
ing to  the  degree  of  its  virulence,  are  acute  septicaemia, 
spreading  inflammatory  exudations,  and  circumscribed 
abscesses.  All  three  of  these  conditions  may  sometimes 
be  produced  by  inoculating  the  same  cultures  into  rabbits 
in  varying  amounts. 

12* 


262  BACTERIOLOGY. 

Rabbits,  mice,  guinea-pigs,  dogs,  rats,  cats,  and  sheep 
are  susceptible  to  infection  by  this  organism.  Chickens 
and  pigeons  are  insusceptible.  Young  animals,  as  a 
rule,  are  more  easily  infected  than  old  ones.  Rabbits 
and  mice  are  the  most  susceptible  of  the  animals  used 
for  experimental  purposes,  and  in  testing  the  virulence 
of  a  culture  it  is  well  to  inoculate  one  of  each,  for  with 
the  same  cultures  it  sometimes  occurs  that  it  may  be 
virulent  for  mice  and  not  for  rabbits,  and  vice  versa. 

If  the  culture  is  virulent,  intra-vascular  or  iutra- 
peritoneal  injections  into  rabbits  may  produce  rapid  and 
fatal  septicaemia,  while  subcutaneous  inoculation  of  the 
same  material  may  result  in  only  a  localized  inflamma- 
tory process.  On  the  other  hand,  subcutaneous  inocula- 
tion of  less  virulent  cultures  may  produce  a  local  process, 
while  intra-venous  inoculation  may  be  without  result. 
This  organism  is  the  cause  of  a  number  of  pathological 
conditions  in  human  beings  that  have  not  hitherto  been 
considered  as  related  to  one  another  etiologically.  It  is 
always  present  in  the  inflammed  area  of  the  lung  in 
acute  fibrinous  or  lobar  pneumonia;  it  is  known  to  cause 
acute  cerebro-spinal  meningitis,  endo-  and  pericarditis, 
certain  forms  of  pleuritis,  arthritis  and  peri-arthritis, 
and  otitis  media. 

6.   SEPTICAEMIA   CAUSED   BY   THE   MICBOCOCCUS 
TETBAGENUS. 

Should  the  death  of  the  animal  not  occur  within  the 
first  twenty-eight  to  thirty  hours  after  inoculation,  but 
be  postponed  until  between  the  fourth  and  the  eighth  day, 
it  may  occur  as  a  result  of  invasion  of  the  tissues  by  the 
organism  now  to  be  described,  viz.,  the  micrococcus  tetra- 
genus. 


MICROCOCCUS  TETRAGENUS. .  263 

This  organism  was  discovered  by  Gaffky  and  was 
subsequently  described  by  Koch  in  the  account  of  his 
experiments  upon  tuberculosis.  It  is  often  present  in 
the  saliva  of  healthy  individuals  and  is  commonly 
present  in  the  sputum  of  tuberculous  patients.  Koch 
found  it  very  frequently  in  the  lung  cavity  of  phthisical 
patients.  It,  however,  plays  no  part  in  the  etiology  of 
tuberculosis. 

It  is  a  small  round  coccus  of  about  1  //  transverse 
diameter.  It  is  seen  as  single  cells,  joined  in  pairs  and 
in  threes,  but  its  most  conspicuous  grouping  is  in  fours, 
from  which  arrangement  it  takes  its  name.  In  prepa- 
rations made  from  cultures  of  this  organism  it  is  not 
rare  to  find,  here  and  there,  single  bodies  which  are 
much  larger  than  the  other  individuals  in  the  field. 
Close  inspection  reveals  them  to  be  cells  in  the  initial 
stage  of  division  into  twos  and  fours.  A  peculiarity  of 
this  organism  is  that  the  cells  are  seen  to  be  bound  to- 
gether by  a  transparent  gelatinous  substance. 

When  cultivated  artificially  it  grows  very  slowly. 

Upon  gelatin  plates  the  colonies  appear  as  round, 
sharply  circumscribed,  puuctiform  masses  which  are 
slightly  elevated  above  the  surface  of  the  surrounding 
medium.  Under  a  low  magnifying  power  they  are  seen 
to  be  slightly  granular  and  present  a  more  or  less  glassy 
lustre. 

The  colonies  increase  but  little  in  size  after  the  third 
or  fourth  day.  If  cultivated  as  stab  cultures  in  gelatin 
there  appears  upon  the  surface  at  the  point  of  inocula- 
tion a  circumscribed  white  point,  slightly  elevated  above 
the  surface  and  limited  to  the  immediate  neighborhood 
of  the  point  of  inoculation.  Down  the  needle-track 
the  growth  is  not  continuous,  but  appears  in  isolated, 


264  BACTERIOLOGY. 

round,  dense  white  clumps  or  beads,  which  do  not  de- 
velop beyond  the  size  of  very  small  points. 

It  does  not  liquefy  gelatin. 

Upon  plates  of  nutrient  agar-agar  the  colonies  appear 
as  small,  almost  transparent,  round  points,  which  have 
about  the  same  color  and  appearance  as  a  drop  of  egg 
albumin ;  they  are  very  slightly  opaque.  They  are 
moist  and  glistening.  They  rarely  develop  to  an  extent 
exceeding  1  to  2  mm.  in  diameter. 

Upon  agar-agar  as  stab  or  slant  cultures,  the  surface 
growth  has  more  or  less  of  a  mucoid  appearance.  It 
is  moist,  glistening,  and  irregularly  outlined.  The  out- 
line of  the  growth  depends  upon  the  moisture  of  the 
agar-agar.  It  is  slightly  elevated  above  the  surface 
of  the  medium. 

In  contradistinction  to  the  gelatin  stab- cultures,  the 
growth  in  agar-agar  is  continuous  along  the  track  of  the 
needle. 

The  growth  on  potato  is  a  thick,  irregular,  slimy- 
looking  patch. 

The  presence  of  the  transparent  gelatinous  substance 
which  is  seen  to  surround  these  organisms  renders  them 
coherent,  so  that  efforts  to  take  up  a  portion  of  a  colony 
from  the  agar-agar  or  potato  cultures  result  usually  in 
drawing  out  fine  silky  threads  consisting  of  organisms 
imbedded  in  this  gelatinous  material. 

The  organism  grows  best  at  from  35°  C.  to  38°  C., 
but  can  be  cultivated  at  the  ordinary  room  temperature 
—about  20°  C. 

The  growth  under  all  conditions  is  slow. 

It  grows  both  in  the  presence  of  and  without  oxygen. 

It  is  not  motile. 

It  stains  readily  with  all  the  ordinary  aniline  dyes. 


MICROCOCCUS  TETRAGENUS.  265 

In  tissues  its  presence  is  readily  demonstrated  by  the 
staining  method  of  Gram. 

The  grouping  into  fours  is  particularly  well  seen  in 
sections  from  the  organs  of  animals  dead  of  this  form 
of  septicaemia. 

In  such  sections  the  organisms  will  always  be  found 
within  the  capillaries. 

Inoculation  into  animals.  To  the  naked  eye  no  alter- 
ation can  be  seen  in  the  organs  of  animals  that  have 
died  as  a  result  of  inoculation  with  the  micrococcus 
tetragenus ;  but  microscopic  examination  of  cover-slip 
preparations  from  the  blood  and  viscera  reveals  the 
presence  of  the  organisms  throughout  the  body — espe- 
cially is  this  true  of  preparations  from  the  spleen. 
White  mice  and  guinea-pigs  are  susceptible  to  the  dis- 
ease. Gray  mice,  dogs,  and  rabbits  are  not  susceptible 
to  this  form  of  septicaemia.  Subsequent  inoculation  of 
healthy  animals  with  a  drop  of  blood,  a  bit  of  tissue, 
or  a  portion  of  a  pure  culture  of  this  organism  from 
the  body  of  an  animal  dead  of  the  disease,  result  in  a 
reproduction  of  the  conditions  found  in  the  dead  animal 
from  which  the  tissues  or  cultures  were  obtained. 

It  sometimes  occurs  that  in  guinea-pigs  which  have 
been  inoculated  with  this  organism  there  result  local 
pus-formations  instead  of  a  general  septicaemia.  The 
organisms  will  then  be  found  in  the  pus-cavity. 


CHAPTER  XVIII. 


Tuberculosis— Microscopic  appearance  of  miliary  tubercles— Encapsula- 
tion of  tuberculous  foci— Diffuse  caseation— Cavity -formation— Primary  infec- 
tion—Modes of  infection -Location  of  the  bacilli  in  the  tissues— Staining 
peculiarities-Organisms  with  which  the  bacillus  tuberculosis  maybe  con- 
founded—Points of  differentiation. 


SHOULD  the  animal  succumb  to  neither  of  the  septic 
processes  just  described,  then  its  death  from  tuberculosis 
may  be  reasonably  expected. 

When  this  disease  is  in  progress,  alterations  in  the 
lymphatic  glands  nearest  the  seat  of  inoculation  may  be 
detected  by  the  touch  in  from  two  to  four  weeks.  They 
will  then  be  found  to  be  enlarged.  Though  not  constant, 
tumefaction  and  subsequent  ulceratiou  at  the  point  of 
inoculation  may  sometimes  be  observed.  Progressive 
emaciation,  loss  of  appetite,  and  difficulty  in  respiration 
point  to  the  existence  of  the  tubercular  process.  Death 
ensues  in  from  four  to  eight  weeks  after  inoculation. 
At  autopsy  either  general  or  local  tuberculosis  may  be 
found.  The  expressions  of  the  tubercular  process  are 
so  manifold  and  in  different  animals  differ  so  widely  the 
one  from  the  other,  that  no  rigid  law  as  to  what  will  ap- 
pear at  autopsy  can  a  priori  be  laid  down. 

The  guinea-pig,  which  is  best  suited  for  this  experi- 
ment, because  of  the  greater  regularity  of  its  suscepti- 
bility to  the  disease  over  that  of  other  animals  usually 
found  in  the  laboratory,  presents,  in  the  main,  changes 
that  are  characterized  by  a  condition  of  coagulation- 
necrosis  and  caseation.  This  is  particularly  the  case 


TUBERCULOSIS.  267 

when  the  infection  is  general,  i.  e.,  when  the  process  is 
of  the  acute  miliary  type.  This  pathological-anatomical 
alteration  is  best  seen  in  the  tissues  of  the  liver  and 
spleen  of  these  animals,  where  the  condition  is  most 
pronounced. 

In  general,  the  tubercular  lesions  can  be  divided  into 
those  of  strictly  focal  character,  i.  e.,  the  miliary  and  the 
conglomerate  tubercles,  and  those  which  are  more  diffuse 
in  their  nature.  The  latter  lesions,  although  of  the  same 
fundamental  nature  as  the  miliary  tubercles,  are  much 
greater  in  extent  and  not  so  sharply  circumscribed. 

These  latter  lesions  play  a  greater  role  in  the  path- 
ology of  the  disease  than  do  the  miliary  nodules,  although 
it  is  to  the  presence  of  the  miliary  nodules  that  the  dis- 
ease owes  its  name. 

At  autopsy  the  pathological  manifestations  of  the  dis- 
ease are  not  infrequently  seen  to  be  confined  to  the  seat 
of  inoculation  and  to  the  neighboring  lymphatic  glands. 
These  tissues  will  then  present  all  the  characteristics  of 
the  tuberculous  process  in  the  stage  of  cheesy  degenera- 
tion. When  the  disease  is  general  the  degree  of  its  ex- 
tension varies.  Sometimes  the  small  gray  nodules — the 
miliary  tubercles — are  only  to  be  seen  with  the  naked 
eye  in  the  tissues  of  the  liver  and  spleen.  Again,  they 
may  invade  the  lungs,  and  commonly  they  are  distrib- 
uted over  the  serous  membranes  of  the  intestines,  the 
lungs,  the  heart,  and  the  brain.  These  simple  gray 
nodules,  as  seen  by  the  naked  eye,  vary  in  size  from 
that  of  a  pin-point  to  that  of  a  hempseed,  and  as  a  rule 
are,  in  this  stage,  the  result  of  the  fusion  of  two  or  more 
smaller  miliary  foci.  Though  the  two  terms,  "  miliary " 
and  "conglomerate/7  exist  for  the  description  of  the 
macroscopic  appearance  of  these  nodules,  yet  it  is  very 


268  BACTERIOLOGY. 

rarely  that  any  condition  other  than  that  due  to  the 
fusion  together  of  several  of  these  minute  foci  can  be 
detected  by  the  naked  eye. 

The  miliary  tubercles  are  of  a  pale  gray  color,  with  a 
white  centre,  are  slightly  elevated  above  the  surface  of 
the  tissue  in  which  they  exist,  and,  as  stated,  vary  con- 
siderably in  dimensions,  usually  appearing  as  points 
which  range  in  size  from  that  of  a  pin-point  to  that  of 
a  pin-head.  They  are  not  only  located  upon  the  surface 
of  the  organs,  but  are  distributed  through  the  depths  of 
the  tissues.  To  the  touch  they  sometimes  present  noth- 
ing characteristic,  but  may  frequently,  when  closely 
packed  together  in  large  numbers,  give  a  mealy  or 
sandy  sensation  to  the  fingers.  Stained  sections  of  these 
miliary  tubercles  present  an  entirely  characteristic  ap- 
pearance, and  the  disease  may  be  diagnosticated  by  these 
histological  changes  alone,  though  the  crucial  test  in 
the  diagnosis  is  the  finding  of  tubercle  bacilli  in  these 
nodules. 

MICROSCOPIC  APPEARANCE  OF  MILIARY  TUBER- 
CLES.— The  simple  miliary  tubercles  under  the  low  mag- 
nifying power  of  the  microscope  present  somewhat  the 
following  appearance  :  There  is  a  central  pale  area,  evi- 
dently composed  of  necrotic  tissue  because  of  its  inca- 
pacity for  taking  up  the  nuclear  stains  commonly 
employed.  Scattered  here  and  there  through  this  ne- 
crotic area  may  be  seen  granular  masses  irregular  in  size 
and  shape;  they  take  up  the  stains  employed,  and  are 
evidently  the  fragments  of  cell-nuclei  in  the  course  of  de- 
struction. Through  the  necrotic  area  may  here  and  there 
be  seen  irregular  lines,  bands,  or  ridges,  the  remains  of 
tissues  not  yet  completely  destroyed  by  the  necrotic  pro- 
cess. Around  the  periphery  of  this  area  may  sometimes 


DIFFUSE  CASSATION.  269 

be  noticed  large  multi -nucleated  cells,  the  nuclei  of  which 
are  arranged  about  the  periphery  of  the  cell  or  grouped 
irregularly  at  its  poles.  The  arrangement  of  these 
nuclei  appears  in  the  sections  sometimes  as  oval,  some- 
times as  somewhat  crescentic  in  its  grouping.  In  the 
tubercles  from  the  human  subject  these  large  " giant- 
eel  Is,"  as  they  are  called,  are  quite  common.  They  are 
much  less  frequent  in  the  tubercular  tissues  from  lower 
animals. 

Round  about  this  central  focus  of  necrosis  is  seen  a 
more  or  less  broad  zone  of  closely  packed  small  round 
and  oval  bodies  which  stain  readily  but  not  homogene- 
ously. They  vary  in  size  and  shape,  and  are  seen  to  be 
imbedded  in  a  delicate  network  of  fibrinous-looking 
tissue. 

This  fibrin-like  network  in  which  these  bodies  lie, 
and  which  is  a  common  accompaniment  of  giant-cell  for- 
mation, is  in  part  composed  of  fibrin,  but  is  in  the  main, 
most  probably,  the  remains  of  the  interstitial  fibrous 
tissue  of  the  part.  This  zone  of  which  we  are  speaking 
is  the  zone  of  so-called  "  granulation  tissue/'  and  con- 
sists of  leucocytes,  granulation  cells,  fibrin,  and  the 
fibrous  remains  of  the  organ ;  the  irregularly  oval, 
granular  bodies  which  take  up  the  staining  are  the 
nuclei  of  these  cells.  The  zone  of  granulation  tissue 
surrounds  the  whole  of  the  tubercular  process,  and  at 
its  periphery  fades  gradually  into  the  healthy  surround- 
ing tissues  or  fuses  with  a  similar  zone  surrounding 
another  tubercular  focus.  This  may  be  taken  as  a 
description  of  the  typical  miliary  tubercle. 

DIFFUSE  CASSATION. — The  diffuse  caseation,  as  said, 
plays  a  more  important  role  in  the  tuberculous  lesion, 
both  in  the  human  and  experimental  forms,  than  does 


270  BACTERIOLOGY. 

the  formation  of  miliary  tubercles.  In  this  a  large 
area  of  tissue  undergoes  the  same  process  of  necrosis 
and  caseatiou  as  the  centre  of  the  miliary  tubercle.  In 
some  tissues  it  is  more  marked  than  in  others.  These 
tissues  are  the  lungs  and  the  lymph-glands.  In  rab- 
bits, particularly,  all  the  changes  in  the  lung  frequently 
come  under  this  head.  When  this  is  the  case  solid 
masses  are  found,  sometimes  as  large  as  a  pea,  or  in- 
volving even  an  entire  lobe  or  the  whole  lung  in  some 
cases.  They  are  of  a  whitish-yellow,  opaque  color,  and 
on  section  are  peculiarly  dry  and  hard.  Entire  lym- 
phatic glands  may  be  changed  in  this  way.  The  con- 
ditions for  this  caseation  of  the  tissues  are  probably 
given  when  a  large  number  of  tubercle  bacilli  enter  the 
tissue  simultaneously  and  a  wide  area  is  involved, 
instead  of  the  small  centre  of  the  miliary  tubercle. 
Necrosis  is  so  rapid  that  time  is  not  given  for  those 
reactive  changes  to  take  place  in  the  tissues  which  result 
in  the  formation  of  the  outer  zone  of  the  miliary  tubercle. 
In  other  instances  the  entire  caseous  area  is  surrounded 
by  a  granulation  zone  similar  to  that  around  the  caseous 
centre  of  the  miliary  tubercles.  It  is  of  special  impor- 
tance to  recognize  the  connection  between  this  diffuse 
caseation  and  the  tubercle  bacillus,  because  until  its 
nature  was  accurately  determined  the  caseous  pneu- 
monia of  the  lungs  formed  the  chief  obstacle  which 
many  encountered  in  recognizing  the  specific  infec- 
tiousness  of  tuberculosis. 

CAVITY-FORMATION. — The  production  of  cavities 
which  form  such  a  prominent  feature  in  human  tuber- 
culosis, particularly  in  the  lungs,  is  due  to  the  softening 
of  the  necrotic,  caseous  masses  or  of  aggregations  of 
miliary  tubercles.  The  material  softens  and  is  expelled, 


ENCAPSULATION  OF  TUBERCULAR  FOCI.     271 

and  a  cavity  remains.  In  the  wall  of  this  cavity  the 
tuberculous  changes  still  proceed,  both  as  diffuse  casea- 
tion  and  formation  of  miliary  tubercles.  The  whole 
cavity,  with  the  reactive  changes  in  the  tissues  of  its 
walls,  may  be  considered  as  representing  a  single  tubercle, 
its  wall  forming  a  tissue  very  analogous  to  the  outer 
zone  of  the  single  tubercle,  the  cavity  itself  corresponding 
to  the  caseous  centre. 

In  animals  used  for  experiment  cavity  formation  of 
this  sort  is  very  rare,  owing  to  the  greater  resistance  of 
the  caseous  tissue.  That  it  is,  however,  possible  to  pro- 
duce in  rabbits  pulmonary  cavities  in  all  physical  re- 
spects similar  to  those  seen  in  the  human  being,  has  been 
most  beautifully  demonstrated  by  Prudden.  He  showed 
that  when  he  had  injected  into  the  trachea  of  rabbits, 
already  aifected  with  tubercular  consolidation  of  the 
lungs,  fluid  cultures  of  the  streptococcus  pyogenes,  the 
result  of  the  mixed  infection  thus  brought  about  was 
cavity  formation  in  eight  out  of  nine  lungs  subjected  to 
the  conditious  of  the  experiment ;  while  in  only  one  out 
of  eleven  did  cavities  form  under  the  influence  of  the 
tubercle  bacillus  alone.1 

In  the  contents  and  in  the  walls  of  tubercular  cavities 
in  man,  bacteria  other  than  the  tubercle  bacillus  are 
found.  It  is  to  the  influence  of  some  of  these,  as  we 
have  seen,  that  diseases  other  than  tuberculosis  may 
sometimes  be  produced  by  the  inoculation  of  animals 
with  the  sputum  from  such  cases. 

ENCAPSULATION  OF  TUBERCULAR  Foci. — It  not 
uncommonly  occurs  that  round  about  a  necrotic  tuber- 


1  Pruddeu  :  Experimental  Phthisis  in  Eabbits,  with  the  Formation  of  Cavi- 
ties, etc.  Transactions  of  the  Association  of  American  Physicians,  1894,  vol. 
ix.  p.  166. 


272  BACTERIOLOGY. 

cular  focus  there  is  formed  a  fibrous  capsule  which  may 
completely  cut  off  the  diseased  from  the  healthy  tissue 
surrounding  it.  Or  a  tubercular  focus  may,  through 
the  resistance  of  the  tissue  in  which  it  is  located,  be 
more  or  less  completely  isolated.  In  this  condition 
the  diseased  foci  may  lie  dormant  for  a  long  time  and 
give  no  evidence  of  their  existence,  until  by  some  in- 
tercurrent  interference  they  are  caused  to  break  through 
their  envelopes.  With  the  passage  of  the  bacilli  or  their 
spores  from  the  central  foci  into  the  vascular  or  lym- 
phatic circulation  the  disease  may  then  become  general. 

It  is  to  some  such  accident  as  this  that  the  sudden 
appearance  of  general  tubercular  infection  in  subjects 
supposed  to  have  recovered  from  the  primary  local 
manifestations  may  often  be  attributed.  The  breaking- 
down  of  old  caseous  lymphatic  glands  is  a  common 
example  of  this  condition. 

PRIMARY  INFECTION. — The  primary  infection  occurs 
through  either  the  vascular  or  lymphatic  circulation. 
Through  these  channels  the  bacilli  gain  access  to  the 
tissues  and  become  lodged  in  the  finer  capillary  ramifi- 
cations or  in  the  more  minute  lymph-spaces.  Here 
they  find  conditions  favorable  to  their  development,  and 
in  the  course  of  their  life  processes  produce  substances  of 
a  chemical  nature  which  act  directly  in  bringing  about  the 
death  of  the  tissues  in  their  immediate  neighborhood. 
This  tissue-death  is  probably  the  very  first  effect  of  the 
bacilli  in  the  body,  and  represents  the  necrotic  centre, 
which  can  always  be  seen  in  even  the  most  minute 
tubercles.  With  the  production  of  this  progressive 
necrosis — for  progressive  it  is,  as  it  continues  as  long 
as  the  bacilli  live  and  continue  to  produce  their  poison- 
ous products — there  is  in  addition  a  reactive  change  in 


MODES  OF  INFECTION.  273 

the  surrounding  tissues,  which  consists  in  the  formation 
of  the  granulation  zone  at  the  outer  margins  of  the 
dying  and  dead  tissue.  This  zone  consists  of  small, 
round  granulation  cells  and  of  leucocytes,  all  of  which 
are  seen  in  the  meshes  of  the  finer  fibrous  tissues  of  the 
part.  At  the  same  time  alterations  are  produced  in  the 
walls  of  the  vessels  of  the  locality  ;  this  tends  to  occlude 
them,  and  thus  the  process  of  tissue-death  is  favored  by 
a  diminution  of  the  amount  of  nutrition  brought  to 
them.  These  changes  may  continue  until  eventually 
conglomerate  tubercles,  widespread  caseation,  or  cavity- 
formation  results  ;  or  from  one  cause  or  another  the  life 
processes  of  the  bacilli  may  be  checked  and  recovery  occur. 

MODES  or  INFECTION. — Experimentally,  tuberculosis 
may  be  produced  in  susceptible  animals  by  subcutaneous 
inoculation ;  by  direct  injection  into  the  circulation ; 
by  injection  into  the  peritoneal  cavity;  by  feeding  of 
tuberculous  material ;  by  the  introduction  of  the  bacilli 
into  the  air-passages,  and  by  inoculation  into  the  an- 
terior chamber  of  the  eye. 

In  the  human  subject  the  most  common  portals  of 
infection  are,  doubtless,  the  air-passages,  the  alimentary 
tract,  and  cutaneous  wounds.  When  introduced  sub- 
cutaneously  the  resulting  process  finds  its  most  pro- 
nounced expression  in  the  lymphatic  system.  The 
growing  bacilli  make  their  way  into  the  lymphatic 
spaces  of  the  loose  cellular  tissue,  are  taken  up  in  the 
lymph  stream  and  deposited  in  the  neighboring  lymph- 
atic glands.  Here  they  may  remain  and  give  rise  to 
no  alteration  further  than  that  seen  in  the  glands  them- 
selves, or  they  may  pass  on  to  neighboring  glands,  and 
eventually  be  disseminated  throughout  the  whole  lymph- 
atic system,  ultimately  reaching  the  vascular  system. 


274  BACTERIOLOGY. 

After  having  gained  access  to  the  bloodvessels,  the 
results  are  the  same  as  those  following  upon  intra- 
vascular  injection  of  the  bacilli,  namely,  general  tuber- 
culosis quickly  follows,  with  the  most  conspicuous  pro- 
duction of  miliary  tubercles  in  the  lungs  and  kidneys, 
less  numerous  in  the  spleen,  liver,  and  bone  marrow. 

When  inhaled  into  the  lungs,  if  conditions  are  favor- 
able, multiplication  of  the  bacilli  quickly  follows.  With 
their  growth  they  are  mechanically  pressed  into  the  tis- 
sues of  the  lungs.  As  multiplication  continues  some  are 
transported  from  the  primary  seat  of  infection  to  healthy 
portions  of  the  lung  tissue,  there  to  give  rise  to  a  further 
production  of  the  tubercular  process. 

In  the  same  way  infection  through  the  alimentary 
tract  is  in  the  main  due  to  mechanical  pressure  of  the 
bacilli  upon  the  walls  of  the  intestines.  Investigation 
has  shown  that  lesions  of  the  intestinal  coats  are  not 
necessary  for  the  entrance  of  tubercle  bacilli  from  the 
intestines  into  the  body.  They  may  be  transported  from 
the  intestinal  tract  into  the  lymphatics  in  the  same  way 
that  the  fat  droplets  of  the  chyle  find  entrance  into  the 
lymphatic  circulation. 

The  evidence  produced  by  Cornet,1  together  with  gen- 
eral statistical  evidence,  points  to  the  lungs  as  the  most 
common  portal  of  natural  infection  for  the  human  be- 
ing. Unlike  most  pathogenic  organisms,  the  tubercle 
bacillus  is  believed  to  have  the  property  of  forming  spores 
within  the  tissues.  These  spores,  which  are  highly  re- 
sistant and  are  not  destroyed  by  drying,  are  thrown  off 
from  the  lungs  in  the  sputum  of  tuberculous  patients  in 
large  numbers,  and  unless  special  precautions  be  taken 

i  Cornet :  Zeit.  fur  Hygiene,  1889,  Bd.  v.,  S.  191. 


LOCATION  OF  THE  BACILLI  IN  THE  TISSUES.    275 

to  prevent  it,  the  sputum  becomes  dried,  is  ground  into 
dust,  and  sets  free  in  the  atmosphere  the  spores  of  tu- 
bercle bacilli  which  came  with  it  from  the  lungs.  The 
frequency  of  pulmonary  tuberculosis  points  to  this  as 
one  of  the  commonest  sources  and  modes  of  infection. 

LOCATION  OF  THE  BACILLI  IN  THE  TISSUES. — The 
bacilli  will  be  found  to  be  most  numerous  in  those  tissues 
which  are  in  the  active  stage  of  the  process. 

In  the  very  initial  stage  of  the  disease  the  bacilli  will 
be  fewer  in  number  than  later.  At  this  time  only  here 
and  there  single  rods  may  be  found ;  later  they  will  be 
more  numerous,  and,  finally,  when  the  process  has  ad- 
vanced to  a  stage  easily  recognizable  by  the  naked  eye, 
they  will  be  found  in  the  granulation  zones  in  clumps 
and  scattered  about  in  large  numbers. 

In  the  central  necrotic  masses,  which  consist  of  cell 
detritus,  it  is  rare  that  the  organisms  can  be  demon- 
strated microscopically.  It  is  at  the  periphery  of  these 
areas  and  in  the  progressing  granular  zone  that  they  are 
most  frequently  to  be  seen. 

This  apparent  absence  of  the  bacilli  from  the  central 
necrotic  area  must  not  be  taken,  however,  as  evidence 
that  this  tissue  does  not  contain  them.  As  bacilli,  they 
are  difficult  to  demonstrate  here  because  the  probabili- 
ties are  that  in  this  locality,  owing  to  conditions  unfa- 
vorable to  their  further  growth,  they  are  in  the  spore 
stage,  a  stage  in  which  it  is  as  yet  impossible,  with  our 
present  methods  of  staining,  to  render  them  visible. 
The  fact  that  this  tissue  is  infective,  and  with  it  the 
disease  can  be  reproduced  in  susceptible  animals,  speaks 
for  the  accuracy  of  this  assumption.  A  conspicuous 
example  of  this  condition  is  seen  in  old  scrofulous 
glands.  These  glands  usually  present  a  slow  process, 


276  BACTERIOLOGY. 

are  commonly  caseous,  and  always  possess  the  property 
of  producing  the  disease  when  introduced  into  the  tis- 
sues of  susceptible  animals,  and  yet  they  are  the  most 
difficult  of  all  tissues  in  which  to  demonstrate  micro- 
scopically the  presence  of  tubercle  bacilli. 

In  tubercles  containing  giant-cells  the  bacilli  can 
usually  be  demonstrated  in  the  granular  contents  of 
these  cells.  Frequently  they  will  be  found  accumu- 
lated at  the  pole  of  the  cell  opposite  to  that  occupied 
by  the  nuclei,  as  if  there  existed  an  antagonism  between 
the  nuclei  and  the  bacilli.  In  some  of  these  cells, 
however,  the  distribution  of  the  bacilli  is  seen  to  be 
irregular,  and  they  will  be  found  scattered  among  the 
nuclei  as  well  as  in  the  necrotic  centre  of  the  cell.  As 
the  number  of  bacilli  in  the  giant-cell  increases  the  cell 
itself  is  ultimately  destroyed. 

Tubercular  tissues  always  contain  the  bacilli  or  their 
spores,  and  are  always  capable  of  reproducing  the  dis- 
ease when  introduced  into  the  body  of  a  susceptible 
animal.  From  the  tissues  of  this  animal  the  bacilli 
may  again  be  obtained  and  cultivated  artificially,  and 
these  cultures  are  capable  of  again  producing  the 
disease  when  further  inoculated.  Thus  the  postulates 
formulated  by  Koch,  which  are  necessary  to  prove  the 
etiological  role  of  an  organism  in  the  production  of  a 
malady,  are  all  fulfilled. 

THE  TUBERCLE  BACILLUS. — Of  the  three  pathogenic 
organisms  liable  to  occur  in  the  sputum  of  a  tuberculous 
subject,  the  tubercle  bacillus  will  give  us  most  difficulty 
in  our  efforts  at  cultivation. 

It  is,  in  the  strict  sense  of  the  word,  a  parasite  and 
finds  conditions  entirely  favorable  to  its  development 
only  in  the  animal  body.  On  ordinary  artificial  media 


PREPARATIONS  OF  CULTURES  FROM  TISSUES.    277 

the  bacilli  taken  directly  from  the  animal  body  grow 
only  very  imperfectly,  or  in  many  cases,  not  at  all. 
From  this  it  seems  probable  that  there  is  a  difference 
in  the  nature  of  individual  tubercle  bacilli — some  ap- 
pearing to  be  capable  only  of  growth  in  the  animal 
tissues,  while  others  are  apparently  possessed  of  the 
power  to  lead  a  limited  saprophytic  existence.  It  may  be, 
therefore,  that  those  bacilli  which  we  obtain  as  artificial 
cultures  from  the  animal  body  are  offsprings  from  the 
more  saprophytic  varieties.  At  best,  one  never  sees 
with  the  tubercle  bacillus  a  saprophytic  condition  in 
any  way  comparable  to  that  possessed  by  many  of  the 
other  organisms  with  which  we  have  to  deal. 

In  efforts  to  cultivate  this  organism  directly  from  the 
tissues  of  the  animal,  the  method  by  which  one  obtains 
the  best  results  is  that  recommended  by  Koch,  viz.,  culti- 
vation upon  blood-serum.  So  strictly  is  this  organism  a 
parasite  that  very  limited  alterations  in  the  conditions 
under  which  it  is  growing  may  result  in  failure  to  suc- 
cessfully study  it.  It  is,  therefore,  necessary  that  the 
injunctions  for  obtaining  it  in  pure  culture  should  be 
carefully  observed. 

PREPARATION  OF  CULTURES  FROM  TISSUES. — 
Under  strictest  antiseptic  precautions,  remove  from  the 
animal  the  tubercular  tissue — the  liver,  spleen,  or  a 
lymphatic  gland  being  preferable.  Place  the  tissue  in 
a  sterilized  Petri  dish  and  dissect  out  with  sterilized 
scissors  and  forceps  the  small  tubercular  nodules.  Place 
each  nodule  upon  the  surface  of  the  blood-serum,  one 
nodule  in  each  tube,  and  with  a  heavy,  sterilized,  looped 
platinum  needle  or  spatula,  rub  it  carefully  over  the 
surface.  It  is  best  to  dissect  away  twenty  to  thirty  such 
tubercles  and  treat  each  in  the  same  way.  Some  of  the 

13 


278  BACTERIOLOGY. 

tubes  will  remain  sterile,  others  may  be  contaminated  by 
outside  organisms  during  the  manipulation,  while  a  few 
may  give  the  result  desired,  viz.,  a  growth  of  the  tubercle 
bacilli  themselves. 

The  blood-serum  upon  which  the  orgauism  is  to  be 
cultivated  should  be  comparatively  freshly  prepared — 
that  is,  should  not  be  dry. 

After  inoculating  the  tubes  they  should  be  carefully 
sealed  up  to  prevent  evaporation  and  consequent  dry- 
ing. This  is  done  by  burning  off  the  superfluous 
overhanging  cotton  plug  in  the  gas-flame,  and  then  im- 
pregnating the  upper  layers  of  the  cotton  with  either 
sealing-wax  or  paraffin  of  a  high  melting-point ;  or  by 
inserting  over  the  burned  end  of  the  cotton  plug  a  soft 
closely-fitting  cork  that  has  been  sterilized  in  the  steam 
sterilizer  just  before  using  (Ghriskey).  This  precaution 
is  necessary  because  of  the  slow  growth  of  the  organism. 
Under  the  most  favorable  conditions  tubercle  bacilli 
directly  from  the  animal  body  show  no  evidence  of 
growth  for  about  twelve  days  after  inoculation  upon 
blood-serum,  and,  as  they  must  be  retained  during  this 
time  at  the  body  temperature — 37.5°  C. — evaporation 
would  take  place  very  rapidly  and  the  medium  would 
become  too  dry  for  their  development. 

If  these  primary  efforts  result  in  the  appearance  of  a 
culture  of  the  bacilli,  further  cultivations  may  be  made 
by  taking  up  a  bit  of  the  colony,  preferably  a  moderately 
large  quantity,  and  transferring  it  to  fresh  serum,  and 
this  in  turn  is  sealed  up  and  retained  at  the  same  tem- 
perature. Once  having  obtained  the  organism  in  pure 
culture,  its  subsequent  cultivation  may  be  conducted 
upon  the  glycerin-agar-agar  mixture — ordinary  neutral 
nutrient  agar-agar  to  which  6  or  7  per  cent,  of  glycerin 


PREPARATION  OF  CULTURES  FROM  TISSUES.     279 

has  been  added.  This  is  a  very  favorable  medium  for 
the  growth  of  this  organism  after  it  has  accommodated 
itself  to  its  saprophy  tic  mode  of  existence,  though  blood- 
serum  is  perhaps  the  best  medium  to  be  employed  in 
obtaining  the  first  generation  of  the  organism  from  the 
tubercular  tissues. 

The  organism  may  be  cultivated  also  on  neutral  milk 
to  which  1  per  cent,  of  agar-agar  has  been  added,  also 
upon  the  surface  of  potato,  and  likewise  in  meat  infusion 
bouillon  containing  6  or  7  per  cent,  of  glycerin. 

Cultures  of  the  tubercle  bacillus  are  characteristic  in 
appearance — after  once  having  seen  them  there  is  but 
little  probability  of  subsequent  mistake. 

They  appear  as  dry  masses,  which  may  develop  upon 
the  surface  of  the  medium  either  as  flat  scales  or  as 
lumps  of  mealy-looking  granules.  They  are  never 
moist,  and  frequently  have  the  appearance  of  coarse 
meal  which  has  been  spread  upon  the  surface  of  the 
medium.  In  the  lower  part  of  the  tube  in  which  they 
are  growing,  i.e.,  that  part  occupied  by  a  few  drops  of 
fluid  which  has  in  part  been  squeezed  from  the  medium 
during  the  process  of  solidification,  and  is  in  part  water 
of  condensation,  the  colonies  may  be  seen  to  float  as  a 
thin  pellicle  upon  the  surface  of  the  fluid. 

The  individuals  making  up  the  growth  adhere  so 
tenaciously  together  that  it  is  with  the  greatest  difficulty 
that  they  can  be  completely  separated.  In  even  the 
oldest  and  dryest  cultures  pulverization  is  impossible. 
The  masses  can  only  be  separated  and  broken  up  by 
grinding  in  a  mortar  with  the  addition  of  some  foreign 
substance,  such  as  very  fine,  sterilized  sand,  dust,  etc. 

The  cultures  are  of  a  dirty-drab  or  brownish-gray 
color  when  seen  on  serum  or  on  glycerin-agar-agar. 


280  BACTERIOLOGY. 

On  potato  they  grow  in  practically  the  same  way, 
though  the  development  is  much  more  limited.  They 
are  here  of  nearly  the  same  color  as  the  potato  on  which 
they  are  growing.  When  cultivated  for  a  time  on  potato 
they  are  said  to  lose  their  pathogenic  properties. 

On  milk-agar-agar  they  are  of  so  nearly  the  same 
color  as  the  medium  that,  unless  they  are  growing  as 
the  mealy-looking  masses,  considerably  elevated  above 
the  surface,  their  presence  is  less  conspicuous  than  when 
on  the  other  media. 

In  bouillon  they  grow  as  a  thin  pellicle  on  the  sur- 
face. This  may  fall  to  the  bottom  of  the  fluid  and  con- 
tinue to  develop,  its  place  on  the  surface  being  taken 
by  a  second  pellicle. 

Under  all  conditions  of  artificial  development  the 
cultures  of  this  organism  are  always  very  dry  and 
brittle  in  appearance,  though  in  truth  the  individuals 
adhere  tenaciously  together  by  a  very  glutinous  sub- 
stance. 

The  tubercle  bacillus  does  not  develop  on  gelatin? 
because  of  the  low  temperature  at  which  this  medium 
must  be  used. 

MICROSCOPIC  APPEARANCE  OF  THE  TUBERCLE 
BACILLUS. — Microscopically  the  organism  itself  is  a 
delicate  rod,  usually  somewhat  beaded  in  its  structure, 
though  rarely  it  is  seen  to  be  homogeneous.  It  is  either 
quite  straight  or  somewhat  curved  or  bent  on  its  long 
axis.  In  some  preparations  involution-forms,  consist- 
ing of  rods  a  little  clubbed  at  one  extremity  or  slightly 
bulging  at  different  points,  may  be  detected.  Branching 
forms  of  this  organism  have  been  described.  It  varies 
in  length — sometimes  being  seen  in  very  short  segments, 
again  much  longer,  though  never  as  long  threads.  On 


STAINING  PECULIARITIES.  281 

an  average,  its  length  is  seen  to  vary  from  2  to  5  JJL.  It 
is  commonly  described  as  being  in  length  about  one- 
fourth  to  one- half  the  diameter  of  a  red  blood-corpuscle. 
It  is  very  slender.  (Fig.  55,  page  256.) 

These  rods  usually  present,  as  has  been  said,  an  ap- 
pearance of  alternate  stained  and  colorless  portions.  It 
is  the  latter  portions  which  are  believed  to  be  the  spores 
of  the  organism,  though,  as  yet  no  absolute  proof  of  this 
opinion  has  been  established. 

At  times  these  colorless  portions  are  seen  to  bulge 
slightly  beyond  the  contour  of  the  rod,  and  in  this  way 
give  to  the  rods  the  beaded  appearance  so  commonly 
ascribed  to  them. 

STAINING  PECULIARITIES. — A  peculiarity  of  this 
organism  is  its  behavior  toward  staining  reagents,  and 
by  this  means  alone  it  may  be  easily  recognized.  The 
tubercle  bacillus  does  not  stain  by  the  ordinary  methods. 
It  possesses  some  peculiarity  in  its  composition  that 
renders  it  more  or  less  proof  against  the  simpler  dyes. 
It  is  therefore  necessary  that  more  energetic  and  pene- 
trating reagents  than  the  ordinary  watery  solutions 
should  be  employed.  Experience  has  taught  us  that 
certain  substances  not  only  increase  the  solubility  of  the 
aniline  coloring  substances,  but  by  their  presence  the 
penetration  of  the  coloring  agents  is  very  much  in- 
creased. Two  of  these  substances  are  aniline  oil  and 
carbolic  acid.  They  are  employed  in  the  solutions  to 
about  the  point  of  saturation.  (For  the  exact  propor- 
tions see  chapter  on  Staining  Reagents.) 

Under  the  influence  of  heat  these  solutions  are  seen 
to  stain  all  bacteria  very  intensely — the  tubercle  bacilli 
as  well  as  the  ordinary  forms.  If  we  subject  our  prep- 
aration, which  may  contain  a  mixture  of  tubercle  bacilli 


282  BACTERIOLOGY. 

and  other  forms,  to  the  action  of  decolorizing  agents,  an- 
other peculiarity  of  the  tubercle  bacillus  will  be  observed. 
While  all  other  organisms  in  the  preparation  will  give 
up  their  color  and  become  invisible,  the  tubercle  bacillus 
retains  it  with  marked  tenacity.  It  stains  with  great 
difficulty,  but  once  stained  it  retains  the  color  even  under 
the  influence  of  strong  decolorizing  agents. 

ORGANISMS   WITH    WHICH   THE   BACILLUS   TUBERCU- 
LOSIS  MAY   BE   CONFUSED. 

DIFFERENTIAL  DIAGNOSIS. — While  its  peculiar 
micro-chemical  reaction  is  usually  considered  to  be  diag- 
nostic of  the  bacillus  tuberculosis,  it  is  well  to  remember 
that  there  are  at  least  three  other  species  of  bacilli  which, 
when  similarly  treated,  react  in  the  same  way.  It  is  of 
importance  to  bear  this  point  in  mind,  particularly  in 
the  microscopic  examination  of  urine  and  pathological 
secretions  from  the  genito-urinary  tract  and  from  the 
rectum,  for  of  the  three  species  two  are  frequently  found 
in  these  localities,  viz.,  the  so-called  smegma  bacillus, 
located  in  the  smegma  and  often  seen  beneath  the  pre- 
puce and  upon  the  vulva,  both  normally  and  in  disease, 
and  the  bacillus  of  syphilis,  described  by  Lustgarten  as 
contained  in  syphilitic  manifestations,  particularly  in 
primary  sores.  The  third  organism  of  this  group — the 
bacillus  of  leprosy — because  of  its  rarity,  is  not  so  likely 
to  cause  error  in  the  diagnosis  of  troubles  occurring  in 
these  localities. 

According  to  Hueppe,  the  differential  diagnosis  be- 
tween the  four  organisms  depends  upon  the  following 
reactions :  When  stained  by  the  carbol-fuchsin  method 
commonly  employed  in  staining  the  tubercle  bacillus  the 


DIFFERENTIAL  DIAGNOSIS.  283 

syphilis  bacillus  becomes  almost  instantly  decolorized 
by  treatment  with  mineral  acids,  particularly  sulphuric 
acid,  whereas  the  smegma  bacillus  resists  such  treatment 
for  a  much  longer  time,  and  the  lepra  and  tubercle 
bacillus  for  a  still  longer  time.  On  the  other  hand,  if 
decolor! zation  is  practised  with  alcohol,  instead  of  acids, 
the  smegma  bacillus  is  the  first  to  lose  its  color.  The 
bacillus  tuberculosis  and  the  bacillus  of  leprosy  are  con- 
spicuously retentive  of  their  color  even  after  treatment 
with  both  acids  and  alcohol. 

To  differentiate,  then,  between  the  four  organisms  he 
recommends  the  following  order  of  procedure,  based  on 
the  above  reactions : 

1.  Treat  the  preparation,  stained  with  carbol-fuchsin, 
with  sulphuric  acid;  the  syphilis  bacillus  becomes  decol- 
orized, the  reaction  being  almost  instantaneous. 

2.  If  it  is  not  at  once  decolorized,  treat  with  alcohol ; 
if  it  is  the  smegma  bacillus  this  will  rob  it  of  its  color. 

3.  If  it  is  still  not  decolorized  it  is  either  the  lepra  or 
tubercle  bacillus. 

The  differential  diagnosis  between  the  last  two  organ- 
isms is  less  satisfactory ;  they  both  take  on  the  same 
stains  and  both  retain  them  or  give  them  up  under 
treatment  with  the  same  decolorizers.  The  results  of 
investigations,  however,  indicate  differences  in  the  rate 
of  staining  and  decolorization,  and  it  is  accepted  by 
many  of  those  who  have  compared  the  two  organisms 
that  the  lepra  bacillus  takes  up  staining  very  much  more 
readily  than  does  the  tubercle  bacillus,  often  staining 
perfectly  by  an  exposure  of  only  a  few  minutes  to  cold 
watery  solutions  of  the  dyes,  but  when  once  stained  it 
retains  its  color  much  more  tenaciously  when  acted  upon 
by  decolorizing  agents  than  does  the  latter  organism. 


284  BACTERIOLOGY. 

According  to  Baumgarten,  the  lepra  bacillus  is  stained 
by  an  exposure  of  six  to  seven  minutes  to  a  cold,  satu- 
rated watery  solution  of  fuchsin,  and  retains  the  stain 
when  subsequently  treated  with  acid  alcohol  (nitric  acid, 
1  part;  alcohol,  10  parts).  By  similar  treatment  for 
the  same  length  of  time  the  bacillus  tuberculosis  does 
not  ordinarily  become  stained. 

These  points,  particularly  what  has  been  said  with 
reference  to  the  smegma  bacillus  and  the  bacillus  of 
syphilis,  are  of  much  practical  importance  and  should 
always  be  borne  in  mind  in  connection  with  microscopic 
examination  of  materials  to  which  these  organisms  are 
liable  to  gain  access.  It  is  hardly  necessary  to  say  that  in 
the  examination  of  sputum  and  pathological  fluids  from 
other  parts  of  the  body,  the  tubercle  bacillus  is,  of  the  four 
organisms,  always  the  one  most  commonly  encountered. 

TUBERCULIN. — The  filtered  products  of  growth  from 
old  fluid  cultures  of  the  tubercle  bacillus  represent  what 
is  known  as  tuberculin — a  group  of  proteid  substances 
possessing  most  interesting  properties.  When  injected 
subcutaneously  into  healthy  subjects,  tuberculin  has  no 
effect,  but  when  introduced  into  the  body  of  the  tuber- 
culous person  or  animal,  a  prouounced  systemic  reaction 
results,  consisting  of  sudden  but  temporary  elevation  of 
temperature,  with,  at  the  same  time,  the  occurrence  of 
marked  hyperaBmia  round  about  the  tuberculous  focus,  a 
change  histologically  analogous  to  that  seen  in  the 
primary  stages  of  acute  inflammation.  This  zone  of 
hypersemia,  with  the  coincident  exudation  and  infiltra- 
tion of  cellular  elements,  probably  aids  in  the  isolation 
or  casting  off  of  the  tuberculous  nodule,  the  inflamma- 
tory zone  forming,  so  to  speak,  a  line  of  demarcation 
between  the  diseased  and  healthy  tissue. 


ANIMALS  SUSCEPTIBLE  TO  TUBERCULOSIS.     285 

As  a  curative  agent  for  the  treatment  of  tuberculosis, 
tuberculin  has  not  merited  the  confidence  that  was  at 
first  accorded  to  it.  Its  greatest  field  of  usefulness  is 
now  admitted  to  be  as  an  aid  to  the  diagnosis  of  obscure 
cases,  and  more  particularly  those  occurring  in  cattle, 
where  it  has  proven  itself  to  be  of  inestimable  value  in 
this  particular  application. 

SUSCEPTIBILITY  OF  ANIMALS  TO  TUBERCULOSIS. — 
The  animals  which  are  known  to  be  susceptible  to  the 
tubercular  processes  are  man,  apes,  cattle,  horses,  sheep, 
guinea-pigs,  pigeons,  rabbits,  cats,  and  field  mice. 

White  mice,  dogs,  and  rats  possess  immunity  against 
the  disease. 

We  have  reviewed  the  three  common  pathogenic  organ- 
isms with  which  we  may  come  in  contact  in  the  sputum 
of  tuberculous  individuals.  Occasionally  oth  er  forms 
may  be  present.  The  pyogenic  forms  are  not  rarely 
found,  and  for  some  time  after  diphtheria  the  bacillus 
of  Lceffler  is  demonstrable  in  the  pharynx,  so  that  it 
too  may  be  present  under  exceptional  circumstances. 
These  latter  organisms  will  be  described  under  their 
proper  heads. 

From  time  to  time  fowls  are  known  to  suffer  from  a 
form  of  tuberculosis  that  is  in  many  respects  similar  to 
human  tuberculosis  both  as  regards  pathological  lesions 
and  etiology.  The  bacillus  causing  the  disease,  while 
very  much  like  the  genuine  bacillus  tuberculosis  mor- 
phologically, differs  from  it  in  cultural  peculiarities ;  in 
its  inability  to  produce  general  tuberculosis  in  rabbits 
and  guinea-pigs ;  in  its  growth  into  long  branched 
forms  at  45°  to  50°  C. ;  and  in  its  never  having  been 
detected  in  human  or  mammalian  tuberculosis. 

Anatomical  lesions  very  suggestive  of  those  produced 
13* 


286  BACTERIOLOGY. 

by  bacillus  tuberculosis  have  also  from  time  to  time 
been  observed  in  certain  rodents.  They  do  not  appear 
to  be  of  specific  nature  as  regards  etiology  for  the  reason 
that  different  authors  have  described  different  species  of 
bacilli  as  the  causative  agents.  The  disease  suggests 
tuberculosis  only  by  the  more  superficial  character  of  its 
lesions,  for  in  no  instance  have  the  organisms  detected 
been  in  any  way  similar  to  the  genuine  bacillus  tubercu- 
losis. These  affections  pass  usually  under  the  name 
pseudo-tuberculosis. 


CHAPTER   XIX. 


Glanders— Characteristics  of  the  disease— Histological  structure  of  the 
glanders  nodule— Susceptibility  of  different  animals  to  glanders— The  ba- 
cillus of  glanders ;  its  morphological  and  cultural  peculiarities— Diagnosis  of 
glanders. 


SYNONYMS  :  Rotz  (G-er.),  Morve  (Fr.). 

Though  most  commonly  seen  in  the  horse  and  ass, 
glanders  is  not  rarely  met  with  in  other  animals,  and  is 
occasionally  encountered  in  man.  When  occurring 
spontaneously  in  the  horse  its  primary  seat  is  usually 
upon  the  mucous  membrane  of  the  nostrils.  It  appears 
in  the  form  of  small  gray  nodules,  about  which  the 
membrane  is  congested  and  swollen.  These  nodules 
ultimately  coalesce  to  form  ulcers.  There  is  a  profuse 
slimy  discharge  from  the  nostrils  during  the  course  of 
the  disease.  It  may  extend  from  its  primary  seat  in 
the  nose  to  the  mouth,  larynx,  trachea,  and  ultimately 
to  the  lungs.  Its  secondary  manifestations  are  observed 
along  the  lymphatics  that  communicate  with  the  primary 
focus,  in  the  lymphatic  glands,  and  as  metastatic  foci  in 
the  internal  organs.  Less  frequently  the  disease  is  seen 
to  begin  in  the  skin,  particularly  in  the  region  of  the 
neck  and  breast.  When  in  this  locality  the  subcuta- 
neous lymphatics  become  involved,  and  are  converted 
into  indurated,  knotty  cords,  easily  discernible  from 
without. 

When  occurring  in  man  it  is  usually  in  individuals 
who  have  been  in  attendance  upon  animals  affected  with 


288  BACTERIOLOGY. 

the  disease.  It  may  occur  upon  the  mucous  membrane 
of  the  nares,  but  its  most  conspicuous  expressions  are  in 
the  skin  and  muscles,  where  appear  abscesses,  phleg- 
mons, erysipelas-like  inflammations,  and  local  necrosis 
closely  resembling  carbuncles.  Metastases  to  the  lungs, 
kidneys,  and  testicles,  as  in  the  horse,  may  also  be  seen. 

When  occurring  upon  the  mucous  membrane  glanders 
is  characterized  by  the  presence  of  small  gray  nodules 
about  as  large  as  a  pin-head,  that  closely  resemble  rnil- 
iary  tubercles  in  their  naked-eye  appearance.  These 
consist  histologically  of  granulation  tissue,  i.  e.,  of  small 
round  cells,  very  similar  to  proliferating  leucocytes,  ot 
some  lymph-cells,  and,  in  the  earliest  stages,  of  a  small 
proportion  of  necrotic  tissue.  As  they  grow  older,  and 
the  process  advances,  there  is  a  tendency  toward  central 
necrosis,  with  the  ultimate  formation  of  a  soft,  yellow, 
creamy,  pus-like  material.  Though  strikingly  like  mil- 
iary  tubercles  in  certain  respects  in  the  early  stages,  there 
are,  nevertheless,  decided  points  of  difference  between 
them. 

The  round-cell  infiltration  of  the  glanders  nodules 
consists  essentially  of  polynuclear  leucocytes,  while  that 
of  the  miliary  tubercle  partakes  more  of  the  nature  of  a 
lymphocytic  infiltration ;  in  the  later  stages  of  the  pro- 
cess the  glanders  nodule  breaks  down  into  a  soft  creamy 
matter,  very  analogous  to  ordinary  pus,  while  in  the 
later  stages  of  the  miliary  tubercle  the  tendency  is  toward 
an  amalgamation  of  its  histological  constituents,  and 
ultimately  to  necrosis  with  caseation.  The  giant-cell 
formation  common  to  tuberculosis  is  never  seen  in  the 
glanders  nodule.  As  Baumgarten  aptly  puts  it :  "The 
pathological  manifestations  of  glanders,  from  the  histo- 
logical aspect,  stand  midway  between  the  acute  purulent 


THE  BACILLUS  OF  GLANDERS.  289 

and  the  chronic  inflammatory  processes."1  Evidently 
these  differences  are  only  to  be  explained  by  differences 
in  the  nature  of  the  causes  that  underlie  the  several 
affections.  We  have  studied  the  characteristics  of  the 
bacillus  tuberculosis  ;  we  shall  now  take  up  the  bacillus 
of  glanders  and  note  the  striking  differences  between 
them. 

THE  BACILLUS  OF  GLANDERS  (bacillus  mallei). — In 
1882  Loeffler  and  Schiitz  discovered  in  the  diseased  tis- 
sues of  animals  suffering  from  glanders  a  bacillus  that, 
when  isolated  in  pure  culture  and  inoculated  into  sus- 
ceptible animals,  possessed  the  property  of  reproducing 
the  disease  with  all  its  clinical  and  pathological  manifes- 
tations. It  is  therefore  the  cause  of  the  disease. 

FIG.  57. 
/       —   << 


Bacillus  of  glanders  (bacillus  mallei). 

It  is  a  short  rod,  with  rounded  or  slightly  pointed 
ends,  that  usually  takes  up  the  staining  somewhat  irreg- 
ularly. (See  Fig.  57.)  When  examined  in  stained  prep- 
arations its  continuity  is  marked  by  alternating  darkly 
and  lightly  stained  areas.  It  is  usually  seen  as  a  single 
rod,  but  may  occur  in  pairs,  and  less  frequently  in  longer 
filaments. 

1  For  &  further  discussion  of  the  pathology  and  pathogenesis  of  this  disease 
see  Lehrbuch  der  pathologischen  Mykologie,  by  Baumgarten,  1890. 


290  BACTERIOLOGY. 

The  question  as  to  its  spore-forming  property  is  still 
an  open  one,  though  the  weight  of  evidence  is  in  oppo- 
sition to  the  opinion  that  it  possesses  this  peculiarity. 
Certain  observers  claim  to  have  demonstrated  spores  in 
the  bacilli  by  particular  methods  of  staining,  but  this 
statement  can  have  but  little  weight  when  compared 
with  the  behavior  of  the  organism  when  subjected  to 
more  conclusive  tests.  For  example,  it  does  not,  at  any 
stage  of  development,  resist  exposure  to  3  per  cent,  car- 
bolic acid  solution  for  longer  than  five  minutes,  nor  to 
1  :  5000  sublimate  solution  for  more  than  two  minutes. 
It  is  destroyed  in  ten  minutes  in  some  experiments,  and 
in  five  in  others,  by  a  temperature  of  55°  C.,  and  when 
dried  it  loses  its  vitality,  according  to  different  observers, 
in  from  thirty  to  forty  days ;  all  of  which  speak  directly 
against  this  being  a  spore-bearing  bacillus. 

It  is  not  motile,  and  does  not,  therefore,  possess 
flagella. 

It  grows  readily  on  the  ordinary  nutrient  media  at 
from  25°  C.  to  38°  C. 

Upon  nutrient  agar-agar,  both  with  and  without 
glycerin,  it  appears  as  a  moist,  opaque,  glazed  layer, 
with  nothing  characteristic  about  it.  This  is  true  both 
for  smear  cultures  and  for  single  colonies. 

Its  growth  on  gelatin  is  much  less  voluminous  than 
on  media  that  can  be  kept  at  higher  temperature,  though 
it  does  grow  on  this  media  at  room  temperature  without 
causing  liquefaction. 

Its  growth  on  blood-serum  is  seen  in  the  form  of  a 
moist,  opaque,  slimy  layer,  inclining  to  a  yellowish  or 
dirty,  brownish-yellow  tinge.  It  does  not  liquefy  the 
serum. 

On  potato  its  growth  is  moderately  rapid,  appearing 


THE  BACILLUS  OF  GLANDERS.  291 

at  the  end  of  from  twenty-four  to  thirty-six  hours  at 
37°  C.  as  a  moist,  amber-yellow,  transparent  deposit 
which  becomes  deeper  in  color  and  denser  in  consistence 
as  growth  progresses.  It  finally  takes  on  a  reddish- 
brown  color,  and  the  potato  about  it  becomes  darkened. 

In  bouillon  it  causes  diffuse  cloudiog,  with  ultimately 
the  formation  of  a  more  or  less  tenacious  or  ropy  sedi- 
ment. 

In  milk  to  which  a  little  litmus  has  been  added,  it 
causes  the  blue  color  to  become  red  or  reddish  in  from 
four  to  five  days,  and  quite  red  after  two  weeks  at  37° 
C.  At  the  same  time  the  milk  is  separated  into  a  firm 
clot  of  casein  and  clear  whey. 

Its  reactions  to  heat  are  very  interesting — at  42°  C. 
it  will  often  grow  for  twenty  days  or  more.  It  will  not 
grow  at  43°  C.,  and  is  killed  by  exposure  to  this  tem- 
perature for  forty-eight  hours.  It  is  killed  in  five  hours 
when  exposed  to  50°  C.,  and  in  five  minutes  by  55°  C. 

It  grows  both  with  and  without  oxygen  ;  it  is  there- 
fore facultative  as  regards  its  relation  to  this  gas. 

On  cover-slips  it  stains  readily  with  all  the  basic 
aniline  dyes,  and,  as  a  rule,  as  stated,  presents  conspicu- 
ous irregularities  in  the  way  that  it  takes  up  the  dyes, 
being  usually  marked  by  deeply  stained  areas  that 
alternate  with  points  at  which  it  either  does  not  stain 
at  all  or  only  slightly. 

The  animals  that  are  susceptible  to  infection  by  this 
organism  are  horses,  asses,  field  mice,  guinea-pigs,  and 
cats.  Baumgarten  records  cases  of  infection  in  lions 
and  tigers  that  have  been  fed,  in  menageries,  with  flesh 
from  horses  affected  with  the  disease.  Rabbits  are  but 
slightly  susceptible  ;  dogs  and  sheep  still  less  so.  Man 
is  susceptible,  and  infection  not  rarely  terminates  fatally. 


292  BACTERIOLOGY. 

White  mice,  common  gray  house-mice,  rats,  cattle,  and 
hogs  are  insusceptible. 

INOCULATION  EXPERIMENTS. — The  most  favorable 
animal  upon  which  to  study  the  pathogenic  properties 
of  this  organism  in  the  laboratory  is  the  common  field 
mouse.  When  inoculated  subcutaneously  with  a  small 
portion  of  a  pure  culture  of  the  glanders  bacillus  death 
ensues  in  about  seventy-two  hours.  The  most  con- 
spicuous tissue  changes  will  be  enlargement  of  the  spleen, 
which  is  at  the  same  time  almost  constantly  studded 
with  minute  gray  nodules,  the  typical  glanders  nodule. 
They  are  rarely  present  in  the  lungs,  but  may  frequently 
be  seen  in  the  liver.  From  these  nodules  the  glanders 
bacillus  may  be  obtained  in  pure  culture.  With  the 
exception  of  the  characteristic  nodule,  the  disease  as 
seen  in  this  animal,  presents  none  of  the  charcteristics 
that  it  displays  in  the  horse  and  ass.  The  clinical  and 
pathological  manifestations  resulting  from  inoculation  of 
guinea-pigs  are  much  more  characteristic.  The  animal 
lives  usually  from  six  to  eight  weeks  after  inoculation, 
and  in  this  time  becomes  affected  with  a  group  of  most 
interesting  and  peculiar  pathological  processes.  The 
specific  inflammatory  condition  of  the  mucous  mem- 
brane of  the  nostrils  is  almost  always  present.  The 
joints  become  swollen  and  infiltrated  to  such  an  extent 
as  often  to  interfere  with  the  use  of  the  legs.  In  male 
animals  the  testicles  become  enormously  distended  with 
pus,  and  on  closer  examination  a  true  orchitis  and 
epididymitis  is  seen  to  be  present.  The  internal  organs, 
particularly  the  lungs,  kidneys,  spleen,  and  liver,  are 
usually  the  seat  of  the  nodular  formations  character- 
istic of  the  disease.  From  all  of  these  disease  foci  the 
bacillus  causing  them  can  be  isolated  in  pure  culture. 


S1AINING  IN  TISSUES.  293 

STAINING  IN  TISSUES. — Though  always  present  in 
the  diseased  tissues,  considerable  trouble  is  usually  ex- 
perienced in  demonstrating  the  bacteria  by  staining 
methods.  The  difficulty  lies  in  the  fact  that  the  bacilli 
are  very  easily  decolorized,  and  in  tissues  stained  by 
the  ordinary  processes  are  robbed  of  their  color  even  by 
the  alcohol  with  which  the  tissue  is  rinsed  out  and  de- 
hydrated. If  we  will  remember  not  to  employ  concen- 
trated stainings,  and  not  to  expose  the  sections  to  the 
stainings  for  too  long  a  time,  but  little  treatment  with 
decolorizing  agents  is  necessary,  and  very  satisfactory 
preparations  will  be  obtained.  A  number  of  good 
methods  have  been  suggested  for  staining  the  glanders 
bacilli  in  tissues,  and  if  what  has  been  said  will  be  borne 
in  mind  no  difficulty  should  be  experienced. 

Two  satisfactory  methods  that  we  have  used  for  this 
purpose,  though  perhaps  no  better  than  some  of  the 
others,  are  as  follows  : 

(a)  Transfer  the  sections  from  alcohol  to  distilled 
water.  This  lessens  the  violence  with  which  the  stain- 
ing subsequently  takes  hold  of  the  tissues,  by  diminish- 
ing the  activity  of  the  diffusion  that  would  occur  if 
they  were  placed  from  alcohol  into  watery  solutions  of 
the  dyes.  Transfer  from  distilled  water  to  the  slide, 
absorb  all  water  with  blotting-paper,  and  stain  with 
two  or  three  drops  of 

Carbolic  fuchsin 10  c.c. 

Distilled  water     .......    100  c.c. 

for  thirty  minutes ;  absorb  all  superfluous  staining  with 
blotting-paper,  and  wash  the  section  three  times  with 
0.3  per  cent,  acetic  acid,  not  allowing  the  acid  to  act  for 
more  than  ten  seconds  each  time.  Remove  all  acid  from 
the  section  by  carefully  washing  in  distilled  water  ;  ab- 


294  BACTERIOLOGY. 

sorb  all  water  by  gentle  pressure  with  blotting-paper, 
and  finally,  at  very  moderate  heat,  or  with  a  small  bel- 
lows (Kiihne),  dry  the  section  completely  on  the  slide. 

When  dried  clear  up  in  xylol,  and  mount  in  xylol 
balsam. 

(6)  Transfer  sections  from  alcohol  to  distilled  water ; 
from  water  to  the  dilute  fuchsin  solution,  and  gently 
warm  (about  50°  C.)  for  fifteen  to  twenty  minutes. 
Transfer  sections  from  the  staining  solution  to  the  slide, 
absorb  all  superfluous  staining  with  blotting-paper,  and 
then  treat  them  with  1  per  cent,  acetic  acid  from  one- 
half  to  three-quarters  of  a  minute.  Remove  all  trace 
of  acid  with  distilled  water,  absorb  all  water  by  gentle 
pressure  with  blotting-paper,  and  then  treat  the  sections 
with  absolute  alcohol  by  allowing  it  to  flow  over  them 
drop  by  drop.  For  small  sections  three  or  four  drops 
are  sufficient.  Under  no  circumstances  should  the 
alcohol  be  allowed  to  act  for  more  than  one-quarter  of  a 
minute.  Clear  up  in  xylol  and  mount  in  xylol  balsam. 

In  method  b  the  tissues  are  better  preserved  than  in 
a,  where  they  are  dried. 

Very  good  preparations  are  also  obtained  by  the  use 
of  Lreffler's  alkaline  methylene-blue,  if  care  be  taken 
not  to  stain  for  too  long  a  time  or  to  decolorize  with 
alcohol  too  energetically. 

No  method  of  contrast-stain  for  this  organism  in  tis- 
sues has  been  devised. 

In  properly  stained  tissues  the  bacilli  will  be  found 
most  numerous  in  the  centre  of  the  nodules,  becoming 
fewer  as  we  approach  the  periphery.  They  usually  lie 
between  the  cells,  but  at  times  may  be  seen  almost 
filling  some  of  the  epithelial  cells,  of  which  the  nodule 
contains  more  or  less.  They  are  always  present  in  these 


MALLEIN.  295 

nodules  in  the  tissues ;  they  are  rarely  present  in  the 
blood,  and,  if  so,  in  only  small  numbers. 

DIAGNOSIS  OF  THE  DISEASE  BY  THE  METHOD  OF 
STKAUSS. — From  what  has  been  said  the  diagnosis  of 
glanders  by  routine  bacteriological  methods  is  certain 
and  relatively  easy,  but  requires  time.  In  clinical  work 
it  is  of  great  importance  for  the  diagnosis  to  be  estab- 
lished as  quickly  as  possible.  With  this  in  view  Strauss 
has  devised  a  method  that  has  given  entirely  satisfactory 
results.  It  consists  in  introducing  into  the  peritoneal 
cavity  of  a  male  guinea-pig  a  bit  of  the  suspected  tissue 
or  culture.  If  it  be  from  a  genuine  case  of  glanders  the 
testicles  begin  to  swell  in  about  thirty  hours,  and  as 
this  proceeds  the  skin  over  them  becomes  red  and  shin- 
ing, desquamation  occurs,  evidences  of  pus-formation 
are  seen,  and,  indeed,  the  abscess  (purulent  orchitis) 
often  breaks  through  the  skin.  The  diagnostic  sign  is 
the  tumefaction  of  the  testicles. 

MALLEIN. — The  filtered  products  of  growth  of  the 
glanders  bacillus  in  fluid  media  represent  what  is  known 
as  mallein — a  group  of  compounds  that  bear  to  glanders 
pretty  much  the  same  relation  that  tuberculin  bears  to 
tuberculosis.  It  is  used  with  considerable  success  as  a 
diagnostic  aid  in  detecting  the  existence  or  absence  of 
deep-seated  manifestations  of  the  disease,  the  glanderous 
animal  reacting  in  from  four  to  ten  hours  to  subcuta- 
neous injections  of  mallein,  while  the  animal  not  so 
affected  gives  no  such  reactions. 

It  is  prepared  from  old,  glycerin-bouillon  cultures  of 
the  glanders  bacillus  by  steaming  them  for  several  hours 
in  the  sterilizer,  after  which  they  are  filtered  through 
uuglazed  porcelain. 


CHAPTER    XX. 

Bacillus  dipMherise—Ite  isolation  and  cultivation— Morphological  and  cul- 
tural peculiarities— Pathogenic  properties— Variations  in  virulence. 

FROM  the  gray-white  deposit  on  the  fauces  of  a  diph- 
theritic patient  prepare  a  series  of  cultures  in  the  follow- 
ing way  : 

Have  at  hand  five  or  six  tubes  of  Loeffler's  blood- 
serum  mixture.  (See  article  on  Media.) 

Pass  a  stout  platinum  needle,  which  has  been  steril- 
ized, into  the  membrane  and  twist  it  around  once  or 
twice  or  brush  it  gently  over  the  surface  of  the  mem- 
brane. Without  touching  it  against  anything  else  rub 
it  carefully  over  the  surface  of  one  of  the  serum  tubes ; 
without  sterilizing  it  pass  it  over  the  surface  of  the  sec- 
ond, then  the  third,  fourth,  and  fifth  tube.  Place  these 
tubes  in  the  incubator.  Then  prepare  cover-slips  from 
scrapings  from  the  membrane  on  the  fauces.  If  the  case 
is  true  diphtheria  the  tubes  will  be  ready  for  examina- 
tion on  the  following  day. 

The  reason  that  plates  are  not  made  in  the  regular 
way  in  this  examination  is  that  the  bacillus  of  diphtheria 
develops  much  more  luxuriantly  on  the  serum  mixture, 
from  which  plates  cannot  be  made,  than  it  does  on  the 
media  from  which  they  can  be  made.  The  method  em- 
ployed, however,  insures  a  dilution  in  the  number  of 
organisms  present,  and  this,  in  addition  to  the  fact  that 
the  bacillus  diphtherice  grows  much  more  quickly  on 


BACILLUS  DIPHTHERIA  297 

the  serum  mixture  than  do  other  organisms,  makes  its 
isolation  by  this  method  a  matter  of  but  little  difficulty. 

After  twenty-four  hours  in  the  incubator  the  tubes 
will  present  a  characteristic  appearance.  Their  surfaces 
will  be  marked  at  different  points  by  more  or  less  irreg- 
ular patches  of  a  white  or  cream-colored  growth  which 
is  usually  more  dense  at  the  centre  than  at  its  irregular 
periphery. 

Except  now  and  then,  when  a  few  orange-colored 
colonies  may  be  seen,  these  large  irregular  patches  are 
the  most  conspicuous  objects  on  the  surface  of  the  serum. 
Occasionally,  almost  nothing  else  appears. 

The  cover-slips  made  from  the  membrane  at  the  time 
the  cultures  were  prepared  will  be  found  on  microscopic 
examination  to  present,  in  many  cases,  a  great  variety  of 
organisms,  but  conspicuous  among  them  will  be  noticed 
slightly  curved  bacilli  of  irregular  size  and  outline.  In 
some  cases  they  will  be  more  or  less  clubbed  at  one  or 
both  ends ;  sometimes  they  appear  spindle  in  shape, 
again  as  curved  wedges ;  now  and  then  they  will  be 
seen  irregularly  segmented.  They  are  rarely  or  never 
regular  in  outline.  If  the  preparation  has  been  stained 
with  Loeffler's  alkaline  methylene-blue  solution  many  of 
these  irregular  rods  are  seen  to  be  marked  by  circum- 
scribed points  in  their  protoplasm  which  stain  very  in- 
tensely ;  they  appear  almost  black.  This  irregularity 
in  outline  is  the  morphological  characteristic  of  the 
bacillus  diphtherice  of  Lceffler.  It  must  be  remembered, 
however,  that  the  diagnosis  of  diphtheria  cannot  be 
made  from  the  examination  of  cover-slip  preparations 
alone,  for  there  are  other  organisms  present  in  the  mouth 
cavity,  particularly  in  the  mouths  of  persons  having  de- 
cayed teeth,  the  morphology  of  which  is  so  like  that  of 


298  BA  CTERIOLOG  Y. 

the  bacillus  of  diphtheria  that  they  might  easily  be  mis- 
taken for  that  organism  if  subjected  to  microscopic  ex- 
amination only. 

The  bacillus  diphtherice  of  Loeffler  (its  discoverer)  can 
readily  be  identified  by  its  cultural  peculiarities  in  con- 
nection with  its  pathogenic  activity  when  introduced 
into  tissues  of  susceptible  animals.  In  guinea-pigs  and 
kittens  the  results  of  its  growth  are  histologically  identical 
with  those  found  in  the  bodies  of  human  beings  who 
have  died  of  diphtheria. 

When  studied  in  pure  culture,  its  morphological  and 
cultural  peculiarities  are  as  follows  : 

MORPHOLOGY. — As  obtained  directly  from  the  diph- 
theritic deposit  in  the  throat  of  an  individual  sick  of  the 
disease,  it  is  sometimes  comparatively  regular  in  shape, 
appearing  as  straight  or  slightly  curved  rods  with  more 
or  less  pointed  ends.  More  frequently,  however,  spindle 
and  club  shapes  occur  and  not  rarely  many  of  these  rods 
take  up  the  staining  irregularly ;  in  some  of  them  very 
deeply  stained,  round  or  oval  points  can  be  detected. 

When  cultures  are  examined  microscopically  it  is 
especially  characteristic  to  find  irregular,  bizarre  forms, 
such  as  rods  with  one  or  both  ends  swollen,  and  very 
frequently  rods  broken  at  irregular  intervals  into  short, 
sharply  marked  segments,  either  round,  oval,  or  with 
straight  sides.  Some  forms  stain  uniformly,  others  in 
various  irregular  ways,  the  most  common  being  the  ap- 
pearance of  deeply  stained  granules  in  a  lightly  stained 
bacillus. 

By  a  series  of  studies  upon  this  organism  when  cul- 
tivated under  artificial  conditions,  we  have  found  that 
its  form  depends  very  largely  upon  the  nature  of  its 
environment.  That  is  to  say,  its  morphology  is  always 


MORPHOLOGY.  299 

more  regular,  and  it  is  smaller  on  glycerin  agar-agar 
than  on  other  media  used  for  its  cultivation;  while 
upon  Loeffler's  blood-serum  the  other  extremes  of  devel- 
opment appear ;  here  one  sees,  instead  of  the  very  short, 
spindle,  lancet,  club-shaped,  always  segmented  and 
regularly  staining  forms  as  seen  upon  glycerin-agar-agar, 
long,  irregularly  staining  threads,  that  are  sometimes 
clubbed  and  sometimes  pointed  at  their  extremities. 
They  are  usually  marked  by  areas  that  stain  more  in- 
tensely than  do  the  rest  of  the  rod,  and  at  times  they 
may  be  a  little  swollen  at  the  centre.  These  differences 
are  so  conspicuous  that  microscopic  preparations  from 
cultures  from  the  same  source,  but  cultivated  in  the  one 
case  on  glycerin-agar-agar  and  in  the  other  upon  blood- 
serum,  when  placed  side  by  side  would  hardly  be 
recognized  as  of  the  same  organism,  unless  its  peculiar 
behavior  under  these  circumstances  was  already  known. 

FIG.  58. 


./- 


a 

Bacillus  diphtheria,    a.  Its  morphology  when  cultivated  on  glycerin-agar- 
agar.    b.  Its  morphology  as  seen  in  cultures  on  Loeffler's  blood-serum. 

On  plain  nutrient  agar-agar  (that  is,  nutrient  agar- 
agar  without  glycerin) ;  on  solidified  egg-albumin ;  on 
a  medium  consisting  of  dried  albumin,  as  found  in 
commerce,  dissolved  in  bouillon  (about  10  grammes 
albumin  to  100  c.c.  of  bouillon  containing  1  per  cent, 
of  grape-sugar) ;  in  bouillon  without  glycerin,  and  in 


300  %A  CTERIOLOG  Y. 

bouillon  to  which  a  bit  of  hard-boiled  egg  has  been 
added,  the  morphology  of  the  organism  is  about  inter- 
mediate, in  both  size  and  outline,  between  the  forms  seen 
upon  glycerin-agar-agarand  upon  Loeffier's  blood-serum. 
There  will  appear  about  an  equal  number  of  short 
segmented  and  longer  irregularly  staining  forms,  but  in 
general  the  longest  are  rarely  as  long  as  the  long  forms 
seen  on  blood-serum,  and  throughout  they  are  not  so 
conspicuous  for  the  irregularity  of  their  staining. 

In  cultures  made  upon  two  sets  of  nutrient  agar-agar 
tubes,  differing  only  in  the  fact  that  one  set  contains 
glycerin  to  the  extent  of  6  per  cent.,  while  the  others 
contain  none,  a  noticeable  difference  in  morphology 
can  usually  be  made  out ;  while  the  forms  on  the  gly- 
cerin-agar-agar  cultures  are  throughout  small,  pretty 
regular  in  size,  shape,  and  staining,  those  on  the  plain 
agar-agar  are  larger,  stain  more  irregularly,  vary  more 
in  shape,  and  when  stained  by  Loeffler's  blue  are  not  so 
uniformly  marked  by  pale  transverse  lines  that  give  to 
them  the  appearance  of  being  made  up  of  numerous 
short  segments. 

Though  the  outline  of  this  organism  is  more  regular 
under  some  circumstances  than  others,  it  is  nevertheless 
always  conspicuous  for  its  manifold  variations  in  shape. 

GROWTH  ON  SERUM  MIXTURE. — The  medium  upon 
which  it  grows  most  rapidly  and  luxuriantly,  and  which 
is  best  adapted  for  determining  its  presence  in  diphthe- 
ritic exudation  is,  as  has  been  stated,  the  blood-serum 
mixture  of  Loeffler.  (See  chapter  on  Media.)  On  the 
blood-serum  mixture  the  colonies  of  the  bacillus  diph- 
therise  grow  so  much  more  rapidly  than  the  other 
organisms  usually  present  in  secretions  and  exudations 
in  the  throat  that  at  the  end  of  twenty-four  hours  they 


GELATIN.  301 

are  often  the  only  colonies  that  attract  attention,  and  if 
others  of  similar  size  are  present,  they  are  generally  of 
quite  a  different  aspect.  Its  colonies  are  large,  round, 
elevated,  grayish-white,  or  yellowish  with  a  centre  more 
opaque  than  the  slightly  irregular  periphery.  The  sur- 
face of  the  colony  is  at  first  moist,  but  after  a  day  or  two 
becomes  rather  dry  in  appearance. 

A  blood-serum  tube  studded  over  with  coalescent  or 
scattered  colonies  of  this  organism  is  so  characteristic 
that  one  familiar  with  the  appearance  can  anticipate 
with  tolerable  certainty  the  results  of  microscopic  ex- 
amination. 

GLYCERIN-AGAR-AGAR. — Upon  nutrient  glycerin- 
agar-agar  the  colonies  likewise  present  an  appearance 
that  may  readily  be  recognized.  They  are  in  every  way 
more  delicate  in  their  structure  than  when  on  the  serum 
mixture.  They  appear  at  first,  when  on  the  surface,  as 
very  flat,  almost  transparent,  dry,  non-glistening,  round 
points  which  are  not  elevated  above  the  surface  upon 
which  they  are  growing.  When  slightly  magnified 
they  are  seen  to  be  granular,  and  present  an  irregular 
central  marking  which  is  denser  and  darker  by  trans- 
mitted light  than  the  thin,  delicate  zone  which  surrounds 
it.  As  the  colony  increases  in  size  the  thin  granular 
peripheral  zone  becomes  broader,  is  usually  marked  by 
ridges  or  cracks,  and  its  periphery  is  notched  or  scal- 
loped. (Fig.  59,  o.)  These  colonies  are  always  quite 
dry  in  appearance.  When  deep  down  in  the  agar-agar 
they  are  coarsely  granular.  (Fig.  59,  a.)  They  rarely 
exceed  3  mm.  in  diameter. 

GELATIN. — On  gelatin  the  colonies  develop  much 
more  slowly  than  on  the  other  media  that  can  be 
retained  at  a  higher  temperature.  They  rarely  present 

14 


302  BACTERIOLOGY. 

their  characteristic  appearances  on  gelatin  in  less  than 
seventy-two  hours. 

They  then   appear  as   flat,  dry,  translucent   points, 
usually  round  in  outline. 

FIG.  59. 


Colonies  of  bacillus  diphtherias  on  glycerin-agar-agar.  a.  Colonies  located 
in  the  depths  of  the  medium.  6.  Colonies  just  breaking  out  upon  the  surface 
of  the  medium,  c.  Fully  developed  surface  colony. 

When  magnified  slightly  the  centre  is  seen  to  be 
more  dense  than  the  surrounding  zone  or  zones,  for  they 
are  sometimes  marked  by  a  concentric  arrangement  of 
zones.  The  periphery  is  irregularly  notched.  Like  the 
colonies  seen  on  agar-agar,  they  are  granular,  but  are 
much  more  granular  when  seen  in  the  depths  of  the 
gelatin  than  when  on  its  surface.  On  gelatin  the  col- 
onies rarely  become  very  large;  usually  they  do  not 
reach  a  diameter  of  over  1.5  mm. 

BOUILLON. — In  bouillon  it  usually  grows  in  fine 
clumps,  which  fall  to  the  bottom  of  the  tube,  or  become 
deposited  on  its  sides  without  causing  a  diffuse  clouding 
of  the  bouillon.  There  are  sometimes  exceptions  to  this 
naked-eye  appearance :  the  bouillon  may  appear  dif- 
fusely clouded,  but  if  one  inspects  it  very  closely,  par- 
ticularly if  one  examines  it  microscopically  in  the  form 
of  a  hanging  drop,  the  arrangement  in  clumps  will  still 


STAB  AND  SLANI  CULTUBES.  303 

be  seen,  but  they  are  so  small  as  not  to  be  detectable  by 
the  unaided  eye. 

In  bouillon  which  is  kept  at  a  temperature  of  35°- 
37°  C.  for  a  long  time,  a  soft,  whitish  pellicle  often 
forms  over  a  part  of  the  surface. 

Changes  in  reactions  of  the  bouillon.  The  reaction  of 
the  bouillon  frequently  becomes  at  first  acid,  and,  sub- 
sequently, again  alkaline,  changes  which  can  be  observed 
in  cultivations  in  bouillon  to  which  a  little  rosolic  acid 
has  been  added.  This  play  of  reactions  has  been 
attributed  to  the  primary  fermentation  of  muscle  sugar 
that  is  often  present  in  the  bouillon. 

POTATO. — On  potato  at  a  temperature  of  35°-37°  C. 
its  growth  after  several  days  is  entirely  invisible ;  only 
a  thin,  dry  glaze  appearing  at  the  point  at  which  the 
potato  was  inoculated.  Microscopic  examination  of 
scrapings  from  the  potato,  after  twenty-four  hours  at 
35°-b7°  C.,  reveals  a  decided  increase  in  the  number  of 
individual  organisms  planted. 

STAB  AND  SLANT  CULTURES. — In  stab  and  slant 
cultures  on  both  gelatin  and  glycerin-agar-agar,  the  sur- 
face growth  is  seen  to  predominate  over  that  along  the 
track  of  the  needle  in  the  depths  of  the  media. 

Isolated  colonies  on  the  surface  of  either  of  the  media 
in  this  method  of  cultivation  present  the  same  charac- 
teristics that  have  been  given  for  the  colonies  on  plates. 

The  growth  in  simple  stab  cultures  does  not  extend 
laterally  very  far  beyond  the  point  at  which  the  needle 
entered  the  medium. 

It  is  a  non-motile  organism. 

It  does  not  form  spores. 

It  is  killed  in  ten  minutes  by  a  temperature  of 
58°  C. 


304  BACTERIOLOGY. 

It  grows  at  temperatures  ranging  from  22°  C.  to 
37°  C.,  but  most  luxuriantly  at  the  latter  temperature. 

Its  growth  in  the  presence  of  oxygen  is  more  active 
than  when  this  gas  is  excluded. 

STAINING. — In  cover-slip  preparations  made  either 
from  the  fauces  of  a  diphtheritic  patient  or  from  a  pure 
culture  of  the  organism,  it  is  seen  to  stain  readily  with 
the  ordinary  aniline  dyes.  It  stains  also  by  the  method 
of  Gram,  but  the  best  results  are  those  obtained  by  the 
use  of  Lreffler's  alkaline  methylene-blue  solution ;  this 
brings  out  the  dark  points  in  the  protoplasmic  body  of 
the  bacilli  and  thus  aids  in  their  identification. 

For  the  purpose  of  demonstrating  the  Loeffler  bacillus 
in  sections  of  diphtheritic  membrane,  both  the  Gram 
method  and  the  fibrin  method  of  Weigert  give  excellent 
results. 

PATHOGENIC  PKOPERTIES. — When  inoculated  sub- 
cutaneously  into  the  bodies  of  susceptible  animals  tl:e 
result  is  not  the  production  of  septicaemia,  as  is  seen  to 
follow  the  introduction  into  animals  of  certain  other 
organisms  with  which  we  shall  have  to  deal,  but  the 
bacillus  of  diphtheria  remains  localized  at  the  point  of 
inoculation,  rarely  disseminating  further  than  the  near- 
est lymphatic  glands.  It  develops  at  the  point  in  the 
tissues  at  which  it  is  deposited,  and  during  its  develop- 
ment gives  rise  to  changes  in  the  tissues  which  result 
entirely  from  the  absorption  of  poisonous  albumins 
produced  by  the  bacilli  in  the  course  of  their  develop- 
ment. 


1  Frosch:  Die  Verbreitung  des  Diphtheric-bacillus  im  Korper  des  Menschen. 
Zeit.  fur  Hygiene  und  Infectionskrankheiten,  1893,  Bd.  xiii.  p.  49-52.  Booker : 
Archives  of  Pediatrics,  Aug.  1893 ;  Wright  and  Stokes :  Boston  Med.  and  Surg. 
Journ.,  March  and  April,  1895. 


PATHOGENIC  PROPERTIES.  305 

In  a  certain  number  of  cases1  diphtheria  bacilli  have 
been  found  in  the  blood  and  internal  organs  of  individ- 
uals dead  of  the  disease,  but  all  that  has  been  learned 
from  careful  study  of  the  secondary  manifestations  of 
diphtheria  tends  to  the  opinion  that  they  are  in  no  way 
dependent  upon  the  immediate  presence  of  bacteria,  and 
that  the  occasional  appearance  of  diphtheria  bacilli  in 
the  internal  organs  is  in  all  probability  accidental,  and 
usually  unimportant. 

By  special  methods  of  inoculation1  (the  injection  of 
fluid  cultures  into  the  testicles  of  guinea-pigs)  diphtheria 
bacilli  can  be  caused  to  appear  in  the  omentum,  but  this 
is  purely  an  artificial  manifestation  of  the  disease  and 
one  that  is  probably  never  encountered  in  the  natural 
course  of  events.  Very  rarely  similar  results  follow 
upon  subcutaneous  inoculation. 

If  a  very  minute  portion  of  either  a  solid  or  fluid 
pure  culture  of  this  organism  be  introduced  into  the  sub- 
cutaneous tissues  of  a  guinea-pig  or  kitten,  death  of  the 
animal  ensues  in  from  twenty-four  hours  to  five  days. 
The  usual  changes  are  an  extensive  local  oedema,  with 
more  or  less  hyperemia  and  ecchymosis  at  the  site  of 
inoculation ;  swollen  and  reddened  lymphatic  glands ; 
increased  serous  fluid  in  the  peritoneum,  pleura,  and 
pericardium  ;  enlarged  and  hemorrhagic  ad-renal  bodies; 
occasionally  slightly  swollen  spleen  ;  and  sometimes 
fatty  degeneration  in  the  liver,  kidney,  and  myocar- 
dium. In  guinea-pigs,  especially,  the  liver  often  shows 
numerous  macroscopic  dots  and  lines  on  the  surface  and 
penetrating  the  substance  of  the  organ.  They  vary  in 
size  from  a  pin-point  to  a  pin-head  and  may  be  even 

1  Abbott  and  Ghriskey  :  A  Contribution  to  the  Pathology  of  Experimental 
Diphtheria.    The  Johns  Hopkins  Hospital  Bulletin,  No.  30,  April,  1893. 


306  BACTERIOLOGY. 

larger.  They  are  white,  and  do  not  project  above  the 
surface  of  the  capsule. 

The  bacilli  are  always  to  be  found  at  the  seat  of  in- 
oculation, most  abundant  in  the  grayish-white,  fibrino- 
purulent  exudate.  They  become  fewer  at  a  distance  from 
this,  so  that  the  more  remote  parts  of  the  oedematous 
tissues  do  not  contain  them.  They  are  found  not  only 
free,  but  contained  in  large  number  in  leucocytes,  some 
of  which  have  fragmented  nuclei,  or  have  lost  their 
nuclei.  The  bacilli  within  leucocytes,  as  well  as  some 
outside,  frequently  stain  very  faintly  and  irregularly, 
and  may  appear  disintegrated  and  dead. 

Culture-tubes  inoculated  from  the  blood,  spleen,  liver, 
kidneys,  ad-renal  bodies,  distant  lymphatic  glands,  and 
serous  transudates,  generally  yield  negative  results  ;  and 
negative  results  are  also  obtained  when  these  organs  are 
examined  microscopically  for  the  bacilli. 

Microscopic  examination  of  the  tissues  at  the  seat  of 
inoculation,  as  well  as  of  the  liver,  spleen,  kidneys, 
lymphatic  glands  and  elsewhere,  reveals  the  presence 
of  localized  foci  of  cell  death,  characterized  by  a  peculiar 
fragmentation  of  the  nuclei  of  the  cells  of  these  parts. 

This  destruction  of  nuclei  results  in  the  occurrence 
of  groups  of  irregularly  shaped,  deeply  staining  bodies, 
having  at  some  times  the  appearance  of  particles  of 
dust,  while  again  they  may  be  much  larger.  Some  of 
them  are  tolerably  regular  in  outline,  while  others  are 
irregularly  crescentic,  dumb-bell,  flask-shape,  whetstone- 
shape,  or  bladder-like  in  form.  Occasionally  nuclei 
having  the  appearance  of  being  pinched  or  drawn  out 
can  be  seen.  At  some  points  the  fragments  are  grouped 
into  isolated  masses,  indicating  the  location  of  the  nucleus 
from  the  destruction  of  which  they  originated.  These 


PATHOGENIC  PROPERTIES.  307 

particles  always  stain  much  more  intensely  than  do  the 
normal  nuclei  of  the  part.1 

These  peculiar  alterations,  as  Oertel  has  shown,  in  their 
distribution,  are  characteristic  of  human  diphtheria,  and 
the  demonstration  of  similar  changes  in  animals  inocu- 
lated with  this  organism  is  important  additional  proof 
that  diphtheria  is  caused  by  it. 

An  affection  may  be  produced  by  the  inoculation  of 
certain  animals  that  is  in  all  respects  identical  with  the 
disease  diphtheria  as  it  exists  in  man.  If  one  opens  the 
trachea  of  a  kitten  and  rubs  upon  the  mucous  membrane 
a  small  portion  of  a  pure  culture  of  this  organism,  the 
death  of  the  animal  usually  ensues  in  from  two  to  four 
days.  At  autopsy  the  wound  will  be  found  covered  with 
a  grayish,  adherent,  uecrotic,  distinctly  diphtheritic 
layer.  Around  the  wound  the  subcutaneous  tissues 
will  be  cedematous.  The  lymphatic  glands  at  the  angle 
of  the  jaws  will  be  swollen  and  reddened.  The  mucous 
membrane  of  the  trachea  at  the  point  upon  which  the 
bacilli  were  deposited  will  be  covered  with  a  tolerably 
firm,  grayish-white,  loosely  attached  pseudo-membrane 
in  all  respects  identical  with  the  croupous  membrane  ob- 
served in  the  same  situation  in  cases  of  human  diph- 
theria. In  the  pseudo-membrane  and  in  the  redematous 
fluid  about  the  skin-wound,  bacilli  diphtherice  may  be 
found  both  in  cover-slips  and  in  cultures. 

From  what  we  have  seen — the  localization  of  the 
bacilli  at  the  point  of  inoculation,  their  absence  from 
the  internal  organs,  and  the  changes  brought  about  in 


1  See  "The  Histological  Changes  in  Experimental  Diphtheria,"  also  "  The 
Histological  Lesions  produced  by  the  Toxalburain  of  Diphtheria,"  by  Welch 
and  Flexner.  The  Johns  Hopkins  Hospital  Bulletin,  August,  1891,  and  March, 


308  BACTERIOLOGY. 

the  cellular  elements  of  the  internal  organs — there  is  but 
one  interpretation  for  this  process,  viz.,  that  it  is  due  to 
the  production  of  a  soluble  poison  by  the  bacteria  grow- 
ing at  the  seat  of  inoculation,  which,  gaining  access  to 
the  circulation,  produces  the  changes  that  we  observe  in 
the  tissues  of  the  internal  viscera. 

This  poison  has  been  isolated  from  cultures  of  the 
bacillus  diphtherias,  and  is  found  to  belong,  not  to  the 
crystallizable  ptomaines,  but  to  the  toxic  albumins — 
bodies  which,  in  their  chemical  composition,  are  anal- 
ogous to  the  poison  of  certain  venomous  serpents.  By 
the  introduction  of  this  toxalbumin,  as  it  is  called,  into 
the  tissues  of  guinea-pigs  and  rabbits,  the  same  path- 
ological alterations  may  be  produced  that  we  have 
seen  to  follow  the  result  of  inoculation  with  the  bacilli 
themselves,  except,  perhaps,  the  production  of  false  mem- 
brane. 

Under  the  influence  of  certain  circumstances  with  which 
we  are  not  acquainted  the  bacillus  diphtherias  becomes  dim- 
inished in  virulence  or  may  lose  it  entirely,  so  that  it  is 
no  longer  capable  of  producing  death  of  susceptible  ani- 
mals, but  may  cause  only  a  transient  local  reaction  from 
which  the  animal  entirely  recovers.  Sometimes  this 
reaction  is  so  slight  as  to  be  overlooked,  and  again  care- 
ful search  may  fail  to  reveal  evidence  of  any  reaction 
at  all.  This  exhibition  of  the  extremes  of  its  patho- 
genic properties,  viz.,  death  of  the  animal,  on  the  one 
hand,  and  only  very  slight  local  effects  on  the  other, 
was  at  one  time  thought  to  indicate  the  existence  of  two 
separate  and  distinct  organisms  that  were  alike  in  cultural 
and  morphological  peculiarities,  but  which  differed  in 
their  disease-producing  power.  Further  studies  on  this 
point  have,  however,  shown  that  the  genuine  bacillus 


PATHOGENIC  PROPERTIES.  309 

diphtherice  may  possess  almost  all  grades  in  the  degree 
of  its  virulence,  and  that  absence  of  or  diminution  in 
virulence  can  hardly  serve  to  distinguish  as  separate 
species  these  varieties  that  are  otherwise  alike ;  more- 
over, the  histological  conditions  found  at  the  site  of 
inoculation  in  animals  that  have  not  succumbed,  but  in 
which  only  the  local  reaction  has  appeared,  are  in  most 
cases  characterized  by  the  same  changes  that  are  seen 
at  autopsy  in  animals  in  which  the  inoculation  has  proven 
fatal. 

In  the  course  of  their  observations  upon  a  large 
number  of  cases,  Roux  and  Yersin  found  that  it  was 
not  difficult  to  detect,  in  the  diphtheritic  deposits  of  one 
and  the  same  individual,  bacilli  of  identical  cultural 
and  morphological  peculiarities,  but  of  very  different 
degrees  of  virulence,  and  that  with  the  progress  of  the 
disease  toward  recovery  the  less  virulent  varieties  often 
became  quite  frequent.1 

There  is,  moreover,  a  mild  form  of  diphtheria  affect- 
ing only  the  mucous  membrane  of  the  nares,  known  as 
membranous  rhinitis,  from  which  it  is  very  common  to 
obtain  cultures  in  all  respects  identical  with  those  from 
typical  diphtheria,  save  for  their  inability  to  kill  suscepti- 
ble animals.  On  inoculation,  these  cultures  produce 
only  local  reactions,  but  they  are  characterized  histo- 
logically  by  the  same  tissue  changes  that  follow  inocu- 
lation with  the  fully  virulent  organism. 

Clinically,  membranous  rhinitis  is  never  such  an 
alarming  disease  as  is  laryngeal  or  pharyugeal  diph- 
theria, and,  as  stated,  the  organisms  causing  it  are  often 

1  It  must  not  be  assumed  from  this  that  the  bacilli  lose  their  virulence 
entirely,  or  that  they  all  become  attenuated  with  the  establishment  of  con- 
valescence. 

14* 


310  BACTERIOLOGY. 

of  a  low  degree  of  virulence,  though  they  are,  never- 
theless, genuine  diphtheria  bacilli. 

For  those  organisms  that  are  in  all  respects  identical 
with  the  virulent  bacillus  diphtherice,  save  for  their  in- 
ability to  kill  guinea-pigs,  the  designation  "  pseudo- 
diphtheritic  bacillus  "  is  usually  employed,  but  from 
such  observations  as  those  just  cited  we  are  inclined  to 
the  opinion  that  pseudo-diphtheritic,  as  applied  to  an 
organism  in  all  respects  identical  with  the  genuine 
bacillus,  except  that  it  is  not  fatal  to  susceptible  ani- 
mals, is  a  misnomer,  and  that  it  would  be  more  nearly 
correct  to  designate  this  organism  as  the  attenuated  or 
non-virulent  diphtheritic  bacillus,  reserving  the  term 
"pseudo-diphtheritic"  for  that  organism  or  group  of 
organisms  (for  there  are  probably  several)  that  is  enough 
like  the  diptheria  bacillus  to  attract  attention,  but  is 
distinguishable  from  it  by  certain  morphological  and 
cultural  peculiarities  aside  from  the  question  of  viru- 
lence. 

It  is  a  well-known  fact  that  many  pathogenic  organ- 
isms —  conspicuous  among  these  being  the  micrococcus 
lanceolatusj  the  staphylococcus  pyogenes  aureus,  and 
the  group  of  so-called  hemorrhagic  septica3mia  organ- 
isms —  undergo  marked  variations  in  the  degree  of  their 
pathogenic  properties,  and  yet  these  organisms,  when 
found  either  devoid  of  this  peculiarity,  or  possessing  it 
to  a  diminished  degree,  are  not  designated  as  "pseudo" 
forms,  but  simply  as  the  organisms  themselves,  the  viru- 
lence of  which,  from  various  causes,  has  been  modified. 


.  —  Prepare  cover-slip  preparations  from  the 
mouth-cavity  of  healthy  individuals  and  from  those 
having  decayed  teeth.  Do  they  correspond  in  any  way 


PATHOGENIC  PROPERTIES.  31 1 

with  those  made  from  diphtheria?  Do  the  same  with 
different  forms  of  sore-throat.  Do  the  peculiarities  of 
any  of  the  organisms  suggest  those  of  the  bacillus  of 
diphtheria  ?  Wherein  is  the  difference  ? 

In  cultures  and  cover  slips  made  from  both  diphtheria 
and  from  innocent  sore-throats  are  there  any  organisms 
which  are  almost  constantly  present  ?  Which  are  they, 
and  what  are  their  characteristics  ? 

Which  are  the  predominating  organisms  in  the  an- 
ginas of  scarlet  fever  ? 

Do  these  organisms  simulate,  in  their  cultural  and 
morphological  peculiarities,  any  of  the  different  species 
with  which  you  have  been  working? 


CHAPTER   XXI. 


Typhoid  fever— Study  of  the  organism  concerned  in  its  production— The 
bacterium  coli  commune — Its  resemblance  to  the  bacillus  of  typhoid  fever — 
Its  morphological,  cultural,  and  pathogenic  properties — Its  differentiation 
from  the  bacillus  typhi  abdominalis. 

THE  organism,  discovered  by  Eberth  and  by  Gaff  ky, 
generally  recognized  as  the  etiological  factor  in  the 
production  of  typhoid  fever,  may  be  described  as  fol- 
lows : 

It  is  a  bacillus  about  three  times  as  long  as  it  is  broad, 
with  rounded  ends.  It  may  appear  at  one  time  as  very 
short  ovals,  at  another  time  as  long  threads,  and  both 
forms  may  occur  together.  Its  breadth  remains  toler- 


FlG.  60. 


FIG.  61. 


*T-vm? 


Bacillus  typhi  abdominalis  from 
culture  twenty-four  hours  old,  on 
agar-agar. 


Bacillus  typhi  abdominalis  show- 
ing flagella  stained  by  Loeffler's 
method. 


ably  constant.  Its  morphology  presents  little  that  will 
aid  in  its  identification  (see  Fig.  60).  It  stains  a  trifle 
less  readily  with  the  aniline  dyes  than  do  most  of  the 
other  organisms.  It  is  very  actively  motile,  and  when 


TYPHOID  FEVER.  313 

stained  by  the  special  method  of  Loeffler  (see  this 
method  in  chapter  on  Stainings)  is  seen  to  possess  very 
delicate  locomotive  organs  in  the  form  of  fine,  hair-like 
flagella,  which  are  given  off  in  large  numbers  from  all 
parts  of  its  surface  (see  Fig.  61).  These  flagella  are 
not  seen  in  unstained  preparations,  nor  are  they  ren- 
dered visible  by  the  ordinary  methods  of  staining. 

In  patients  suffering  from  this  disease  it  has  been 
found  during  life  in  the  blood,  urine,  and  faeces,  and  at 
autopsies,  in  the  tissues  of  the  spleen,  liver,  kidneys, 
intestinal  lymphatic  glands,  and  intestines. 

GELATIN  PLATES. — Its  growth,  when  seen  in  the 
depths  of  the  medium,  presents  nothing  characteristic, 
appearing  simply  as  round  or  oval,  finely  granular 
points.  On  the  surface  it  develops  as  very  superficial, 
blue-white  colonies,  with  irregular  borders.  They  are  a 
little  denser  at  the  centre  than  at  the  periphery.  When 
magnified,  the  colonies  present  wrinkles  or  folds,  which 

FIG.  62. 


Colony  of  bacillus  typhi  abdominalis  on  gelatin. 

give  to  them,  in  miniature,  the  appearance  seen  in  the 
relief  maps  made  to  represent  mountainous  districts 
(Fig.  62).  These  colonies  have  sometimes  the  appear- 
ance of  flattened  pellicles  of  glass-wool,  and  usually 
present  more  or  less  of  a  pearl-like  lustre. 

ON  AGAR-AGAR  the  colonies  present  nothing  typical. 


314  BACTERIOLOGY. 

STAB  CULTURES. — In  stab  cultures  the  growth  is 
mostly  on  the  surface,  there  being  only  a  very  limited 
development  down  the  track  made  by  the  needle.  The 
surface  growth  has  the  same  appearance  in  general  as 
that  given  for  the  colonies. 

POTATO. — The  growth  on  potato  is  usually  described 
as  luxuriant  but  invisible,  making  its  presence  evident 
only  by  the  production  of  a  slight  increase  of  moisture 
at  the  inoculated  point,  and  by  a  limited  resistance 
offered  to  the  needle  when  scraped  across  the  track  of 
growth.  While  this  is  true  in  most  cases,  yet  it  cannot 
be  considered  as  constant,  for  at  times  this  organism  is 
seen  to  develop  more  or  less  visibly  on  potato. 

POTATO  GELATIN. — The  growth  is  similar  to  that 
upon  ordinary  nutrient  gelatin. 

MILK. — It  does  not  cause  coagulation  when  grown  in 
sterilized  milk. 

It  does  not  liquefy  gelatin. 

It  grows  both  with  and  without  oxygen. 

It  does  not  produce  indol. 

In  bouillon  it  causes  a  uniform  clouding  of  the 
medium  and  brings  about  a  slightly  acid  reaction. 

It  does  not  grow  rapidly. 

It  does  not  produce  fermentation,  and  on  lactose- 
litmus-agar-agar  grows  as  pale- blue  colonies,  causing  no 
reddening  of  the  surrounding  medium.  In  the  fermen- 
tation tube,  in  glucose  or  lactose  bouillon,  no  evolution 
of  gas  as  a  result  of  fermentation  occurs. 

It  does  not  form  spores.  The  irregularities  of  stain- 
ing so  commonly  seen  in  this  organism  have  in  some 
instances  led  to  the  belief  that  the  pale,. unstained  por- 
tions of  the  bacilli  indicate  the  presence  of  spores,  More 


PRESENCE  IN  TISSUES.  315 

reliable  tests,  however,  have  demonstrated  the  error  of 
this  opinion. 

It  grows  at  any  temperature  between  20°  C.  and  38° 
C.,  though  more  favorably  at  the  latter  point. 

It  is  very  sensitive  to  high  temperatures,  being  killed 
by  an  exposure  often  minutes  to  60°  C.,  and  in  a  much 
shorter  time  to  slightly  higher  temperatures. 

Owing  to  a  tendency  toward  retraction  of  its  proto- 
plasm from  the  cell  envelope  and  the  consequent  pro- 
duction of  vacuoles  in  the  bacilli,  the  staining  of  this 
organism  is  usually  more  or  less  irregular.  At  some 
points  in  a  single  cell  marked  differences  in  the  intensity 
of  the  staining  will  be  seen,  and  here  and  there  areas 
quite  free  from  color  can  commonly  be  detected.  These 
colorless  portions  are  often  so  cleanly  cut  in  outline  as 
to  look  as  if  they  had  been  punched  out  with  a  sharp 
instrument.  (Diagram matically  represented  in  Fig.  63.) 

FIG.  63. 


Diagrammatic  representation  of  retraction  of  protoplasm,  with  production  of 
pale  points,  in  the  bacillus  typhi  abdominalis. 


PRESENCE  IN  TISSUES. — It  is  not  easy  to  demonstrate 
this  organism  in  tissues  unless  it  is  present  in  large 
numbers.  The  manipulations  to  which  the  sections  are 
subjected  in  being  mounted  often  rob  the  bacilli  of  their 
staining,  and  render  them  invisible,  or  nearly  so.  If, 
however,  sections  be  stained  in  the  carbolic-fuchsin  solu- 
tion, either  at  the  ordinary  temperature  of  the  room  or 


316  BACTERIOLOGY. 

at  a  higher  temperature  (40°  to  45°  C.),  then  washed 
out  in  absolute  alcohol,  and  cleared  up  in  xylol  and 
mounted  in  balsam,  the  bacilli  (particularly  if  the  tissue 
be  the  liver  or  spleen)  can  readily  be  detected,  massed 
together  in  their  characteristic  clumps.  If  used  in  the 
same  way,  the  alkaline  methylene-blue  solution  gives 
also  very  satisfactory  results. 

In  searching  for  the  typhoid  bacilli  in  tissues,  their 
mode  of  growth  under  these  circumstances  must  always 
be  borne  in  mind,  otherwise  much  labor  will  be  ex- 
pended in  vain.  In  tissues  the  typhoid  bacilli  do  not 
lie  scattered  about  in  the  same  way  as  do  the  organisms 
in  tissues  from  cases  of  septicaemia ;  they  are  not  regu- 
larly distributed  along  the  course  of  the  capillaries,  but 
are  localized  in  small  clumps  through  the  tissues,  and  it 
is  for  these  clumps,  which  are  easily  detected  under  the 
low-power  objective,  that  one  should  search.  When  the 
section  is  prepared  for  examination,  if  it  be  gone  over 
with  the  low-power  objective,  one  will  notice  at  irregu- 
lar intervals  little  masses  that  look  in  every  respect  like 
particles  of  staining-matter  which  have  been  precipitated 
upon  the  section  at  that  point.  When  these  little  masses 
are  examined  with  a  higher  power  objective  they  will  be 
found  to  consist  of  small  ovals  or  short  rods  so  closely 
packed  together  that  the  individuals  composing  the 
clump  can  often  be  seen  only  at  the  very  periphery  of 
the  mass.  This  is  the  characteristic  appearance  of  the 
typhoid  organism  in  tissues.  The  little  masses  are 
usually  in  the  neighborhood  of  a  capillary. 

RESULT  OF  INOCULATION  INTO  LOWER  ANIMALS. — 
A  great  many  experiments  have  been  made  with  the 
view  of  reproducing  the  pathological  conditions  of  this 
disease,  as  seen  in  man,  in  the  tissues  of  lower  animals, 


INOCULATION  INTO  LOWER  ANIMALS.        317 

but  with  limited  success.  Fatal  results  without  the  ap- 
pearance of  the  typical  pathological  changes  have  fre- 
quently followed  these  attempts,  but  in  most  cases  they 
could  easily  be  traced  to  the  toxic,1  rather  than  to  the 
truly  infective2  action  of  the  materials  introduced  into 
the  animals. 

The  most  successful  efforts  for  the  production  of  the 
typical  typhoid  lesions  in  lower  animals  are  those  re- 
ported by  Cygnseus.  By  the  introduction  of  the  typhoid 
bacilli  into  the  tissues  of  dogs,  rabbits,  and  mice  he  was 
able  to  produce  in  the  small  intestines  conditions  that 
were  histologically  and  to  the  naked  eye  analogous  to 
those  found  in  the  human  subject. 

Of  a  number  of  experiments  made  by  the  writer  with 
the  same  object  in  view,  only  one  positive  result  fol- 
lowed the  introduction  of  typhoid  bacilli  into  the  circu- 
lation of  rabbits.  In  this  case  the  ulcer  in  the  ileum 
was^macroscopically  and  microscopically  identical  with 
those  found  at  autopsy  in  the  small  intestine  of  the 
human  subject  dead  of  this  disease.  The  typhoid  bacilli 
were  not  only  obtained  from  the  spleen  of  the  animal 
by  culture  methods,  but  were  also  demonstrated  micro- 
scopically in  their  characteristic  clumps  in  sections  of 
the  organ. 

In  connection  with  the  inoculation  of  animals  with 
the  bacillus  typhi  abdominalis,  observations  of  a  most 
important  nature  have  been  made  by  Sanarelli3  upon 
the  artificial  induction  of  susceptibility  to  its  pathogenic 
action.  He  found  that  rabbits,  guinea-pigs,  and  mice 

1  Toxic— Poisonous  results  not  necessarily  accompanied  by  the  growth  of 
organisms  throughout  the  tissues. 

2  Infective  or  septic— Poisoning  of  the  tissues  as  a  result  of  the  growth  of 
bacteria  in  them. 

s  Sanarelli :  Annales  de  1'Institut  Pasteur,  1892,  tome  vi. 


318  BACTERIOLOGY. 

could  be  rendered  susceptible  to  infection  by  this  organ- 
ism by  preliminary  injections  into  them  of  the  products  of 
growth  of  certain  saprophytes — proteus  vulgaris,  bacillus 
prodigiosuSj  and  bacterium  coli  commune — and  that  by 
whatever  means  the  animal  was  subsequently  inoculated 
with  fresh  cultures  of  the  typhoid  bacillus,  either  into 
the  circulation  or  into  the  peritoneal  cavity,  death  re- 
sulted in  from  twelve  to  forty-eight  hours,  with  the  most 
conspicuous  pathological  alterations  in  the  digestive  tract, 
and  particularly  in  the  small  intestines.  In  these  cases 
the  infection  is  general  and  the  organisms  may  be  recov- 
ered from  the  blood  and  internal  organs.  It  is  the 
opinion  of  Sanarelli  that  the  toxic  conditions  produced 
by  the  preliminary  injections  of  the  products  of  growth 
of  the  saprophytic  organisms  may  be  considered  as 
analogous  to  a  similar  condition  that  may  occur  in  man 
from  the  absorption  of  abnormal  products  of  fermenta- 
tion from  the  intestinal  canal — an  auto-intoxication  that 
so  reduces  the  resistance  of  the  individual  as  to  render 
him  susceptible  to  infection  by  the  bacillus  of  typhoid 
fever,  should  it  gain  access  to  his  alimentary  tract. 

More  recently  it  was  found  by  Alessi1  that  rats, 
guinea-pigs,  and  rabbits,  when  permitted  to  breathe  the 
gaseous  products  of  decomposition  from  the  contents  of 
a  cesspool,  or  from  other  decomposing  matters,  gradually 
became  susceptible  to  infection  by  the  typhoid  bacillus. 
After  an  exposure  of  from  five  to  seventy-two  days  in 
the  case  of  rats,  seven  to  fifty-eight  days  in  the  case  of 
guinea-pigs,  and  three  to  eighteen  days  in  the  case  of 
rabbits,  the  resistance  of  the  animals  was  so  diminished 
that  inoculation  with  relatively  small  amounts  of  cul- 

1  Alessi :  Centralblatt  fur  Bakteriolgie  u.  Parasitenkunde,  1894,  Bd.  xv.,  No. 
p.  228. 


L**  ^^  ^ 

-       OF  TH€ 

UNIVERSITY 


TOCULATION  INTO  LOWER  ANIMALS.     319 

tures  of  the  typhoid  bacillus  proved  fatal  in  from  twelve 
to  thirty-six  hours.  Autopsies  upon  these  animals  re- 
vealed the  presence  of  hemorrhagic  enteritis,  hypertrophy 
of  Peyer's  patches,  and  enlargement  of  the  spleen.  The 
bacilli  were  found  in  the  blood,  liver,  and  spleen. 

The  importance  of  these  observations  in  their  bearing 
upon  the  etiology  of  typhoid  fever,  if  they  are  demon- 
strated by  subsequent  experiment  to  be  trustworthy,  is 
too  obvious  to  necessitate  emphasis,  and  it  is  greatly  to 
be  desired  that  they  may  not  be  permitted  to  pass 
unnoticed,  but  that  others  interested  may  find  occasion 
to  institute  experiments  in  the  same  direction  with  the 
hope  that  some  light  may  be  shed  upon  the  mooted 
question  concerning  the  influence  of  gaseous  products 
of  decomposition  upon  the  health  of  individuals,  and 
particularly  upon  the  part  played  by  them  in  diminish- 
ing natural  resistance  to  infection.1 

Because  of  the  variations  in  the  morphology  and  cul- 
tural peculiarities  of  this  organism,  and  because  of  the 
difficulty  experienced  in  efforts  to  reproduce  in  lower 
animals  the  conditions  found  in  the  human  subject, 
typhoid  fever  is  bacteriologically  one  of  the  most  un- 
satisfactory of  the  infectious  diseases. 

There  are  a  number  of  other  organisms  which  botani- 
cally  appear  to  be  nearly  related  to  the  typhoid  bacil- 
lus, and  which,  with  our  present  methods  for  studying 
them,  so  closely  simulate  it,  that  the  difficulty  of  identify- 
ing this  organism  is  sometimes  very  great.  In  addition 
to  this,  the  variability  constantly  seen  in  pure  cultures 


1  See  paper  by  the  author  :  "  The  Effects  of  the  Gaseous  Products  of  Decom- 
position upon  the  Health,  aiid  Resistance  to  Infection,  of  Certain  Animals 
that  are  Forced  to  Respire  Them."  Transactions  of  the  Association  of 
American  Physicians  and  Surgeons,  1895,  vol.  x.  p.  16-44. 


320  BACTERIOLOGY. 

of  the  typhoid  bacillus  itself,  in  no  way  renders  the 
task  more  simple. 

For  example,  the  morphology  of  the  typhoid  bacillus 
is  conspicuously  inconstant ;  its  growth  on  potato,  which 
was  formerly  described  as  characteristic,  may,  with  the 
same  organism,  at  one  time  appear  as  the  typical  invisible 
development,  at  another  time  it  may  grow  in  a  way 
easily  to  be  seen  with  the  naked  eye ;  and  the  change  of 
reaction  which  it  is  said  to  produce  in  bouillon  is  some- 
times much  more  intense  than  at  others. 

The  only  properties  possessed  by  it  that  may  be  said 
to  be  constant  are  its  motility,  its  inability  to  cause 
fermentation  of  glucose,  lactose,  or  saccharose,  its 
incapacity  for  coagulating  milk,  the  absence  of  indol- 
production,  and  its  growth  on  gelatin  plates ;  but  there 
are  other  organisms  which  approach  these  same  charac- 
teristics to  a  degree  that  renders  their  differentiation 
from  the  typhoid  organism  often  a  matter  that  requires 
the  careful  application  of  all  these  different  tests. 

These  points  should  be  borne  in  mind  in  the  exami- 
nation of  drinking-water  supposed  to  be  contaminated 
by  typhoid  dejections,  for  the  organisms  which  most 
nearly  approach  the  typhoid  bacillus  in  growth  and 
morphology  are  just  those  organisms  which  would 
appear  in  water  contaminated  from  cesspools,  i.  e.,  the 
organisms  constantly  found  in  the  normal  intestinal 
tract.  Even  in  the  stools  of  typhoid-fever  patients 
the  presence  of  these  normal  inhabitants  of  the  intes- 
tinal tract  renders  the  isolation  of  the  typhoid  organisms 
somewhat  troublesome, 

The  spleen  of  a  patient  dead  of  typhoid  fever  is  the 
safest  place  from  which  to  obtain  cultures  of  this  organ- 
ism for  study.  But  it  must  always  be  remembered  that 


EXPERIMENTS.  321 

the  same  channels  through  which  the  typhoid  bacillus 
gains  access  to  this  viscns  are  likewise  open  to  other 
organisms  present  in  the  intestines,  and  for  this  reason 
the  bacterium  coli  commune,  a  normal  inhabitant  of  the 
colon,  may  also  be  found  in  this  locality. 

NOTE. — Obtain  a  pure  culture  of  typhoid  bacilli,  and 
from  this  make  inoculations  upon  a  series  of  potatoes  of 
different  age  and  from  different  sources.  Do  they  all 
grow  alike  ? 

Before  sterilizing,  render  another  lot  of  potatoes 
slightly  acid  with  a  few  drops  of  very  dilute  acetic  acid ; 
render  others  very  slightly  alkaline  with  dilute  caustic 
soda.  Do  any  differences  in  the  growth  result? 

Make  a  series  of  twelve  tubes  of  peptone  solution  to 
which  rosolic  acid  has  been  added.  Inoculate  them  all 
with  as  near  the  same  amount  of  material  as  possible 
(one  loopful  from  a  bouillon  culture  into  each  tube) ; 
place  them  all  in  the  incubator.  Is  the  color-change, 
as  compared  with  that  of  the  control  tube,  the  same  in  all 
cases  ? 

Compare  the  morphology  of  cultures  of  the  same  age 
on  gelatin,  agar-agar,  and  potato. 

Select  a  culture  in  which  the  vacuolations  are  quite 
marked.  Examine  this  culture  unstained.  Do  the 
organisms  look  as  if  they  contained  spores?  How 
would  you  demonstrate  that  the  vacuolations  are  not 
spores  ? 

Obtain  from  the  normal  faeces  a  pure  culture  of  the 
commonest  organism  present.  Write  a  full  description 
of  it.  Now  make  parallel  cultures  of  this  organism  and 
of  the  typhoid  organism  on  all  the  different  media. 
How  do  they  differ  ?  In  what  respects  are  they  similar? 


322  BACTERIOLOGY. 

BACTERIUM  COLI  COMMUNE  (colon  bacillus ;  bacillus 
Neapolitanus  of  Emmerich). — This  organism  was  dis- 
covered by  Escherich,in  1885,  in  the  intestinal  discharges 
of  milk-fed  infants.  It  has  since  been  demonstrated  to 
be  a  normal  inhabitant  of  the  intestines  of  man  and  of 
certain  domestic  animals  (cattle,  hogs,  dogs). 

For  a  time  after  its  discovery  it  was  considered  of  but 
little  importance  and  attracted  attention  only  because 
of  its  resemblance,  in  certain  respects,  to  the  bacillus  of 
typhoid  fever,  with  which  it  was  occasionally  confounded. 
In  this  particular  it  still  serves  as  a  subject  for  study.  Some 
have  even  gone  so  far  as  to  regard  them  but  as  varieties  of 
one  and  the  same  species,  though  in  thepresent  state  of  our 
knowledge  this  is  certainly  an  assumption  for  which,  as 
yet,  there  are  not  sufficient  grounds.  That  they  possess 
in  common  certain  general  points  of  resemblance  and 
often  approach  one  another  in  some  of  their  biological 
peculiarities  is  true ;  but,  as  we  shall  learn,  they  each 
possess  peculiarities,  which,  when  taken  together,  render 
their  differentiation  from  one  another  a  matter  of  but 
little  difficulty. 

With  the  wider  application  of  bacteriological  methods 
to  the  study  of  pathological  processes  it  was  occasion- 
ally observed  that,  under  favorable  circumstances,  this 
organism  was  disseminated  from  its  normal  habitat  and 
appeared  in  remote  organs,  often  associated  with  diseased 
conditions.  This  was  also,  at  first,  considered  as  of  but 
trifling  moment,  and  its  presence  in  these  localities  was 
usually  explained  as  accidental.  Its  repeated  appear- 
ance, however,  in  different  parts  of  the  body  outside  of 
the  intestines,  and  the  frequency  of  its  association  with 
pathological  conditions,  ultimately  attracted  attention  to 
it,  and  in  consequence  during  the  past  two  or  three  years 


BACTERIUM  COLI  COMMUNE.  323 

a  great  deal  has  been  written  concerning  the  possible 
pathogenic  nature  of  this  organism. 

The  fact  that  it  is  always  with  us  in  most  intimate 
association  with  certain  of  our  life  processes,  together 
with  the  fact  that  it  is  known  to  appear  in  organs  other 
than  that  in  which  it  is  normally  located,  and  that  its 
occurrence  in  diseased  conditions  is  not  rare,  justifies 
the  opinion  that  it  is  one  of  the  most  important  of  the 
micro-organisms  with  which  we  have  to  deal. 

While  not  generally  considered  to  be  a  pathogenic 
organism,  there  is,  nevertheless,  sufficient  evidence  to 
warrant  the  statement  that,  under  favorable  conditions, 
with  which  we  are  not  entirely  familiar,  this  organism 
may  assume  pathogenic  properties  and  that  its  presence 
in  diseased  conditions  is  not  always  to  be  considered  as 
accidental,  though  this  is  frequently  the  case. 

The  morphological  and  cultural  peculiarities  of  the 
bacterium  coli  commune  are  as  follows : 

Morphology.  In  shape  it  is  a  rod  with  rounded  ends, 
sometimes  so  short  as  to  appear  almost  spherical,  while 
again  it  is  seen  as  very  much  longer  threads.  Often 
both  forms  will  be  associated  in  the  same  culture.  It 
may  occur  as  single  cells  or  as  pairs,  joined  end-to-end. 
There  is  nothing  to  be  said  of  its  morphology  that  can 
aid  in  its  identification,  for  in  this  respect  it  simulates 
many  other  organisms.  It  is  usually  said  to  be  motile, 
and  undoubtedly  is  motile  in  the  majority  of  cases,  but 
its  movements  are  so  sluggish  that  a  positive  opinion  is 
often  difficult. 

By  Lceffler's  method  of  staining,  flagella  can  be  dem- 
onstrated, though  not  in  such  numbers  as  are  seen  to 
occur  on  the  typhoid  fever  bacillus. 

It  does  not  form  spores. 


324  BACTERIOLOGY. 

It  grows  both  with  and  without  oxygen. 

On  gelatin.  When  on  the  surface,  its  colonies  appear 
small,  dry,  irregular,  flat,  blue-white  points  that  are 
commonly  somewhat  dentated  at  the  margin.  They  are 
a  trifle  denser  at  the  centre  than  at  the  periphery,  and 
are  often  marked  at  or  near  the  middle  by  an  oval  or 
round  nucleus-like  mass — the  original  colony  from  which 
the  layer  on  the  surface  developed.  When  located  in 
the  depths  of  the  gelatin,  and  examined  with  a  low- 
power  lens,  they  are  at  first  seen  to  be  finely  granular 
and  of  a  very  pale  greenish-yellow  color ;  later  they 
become  denser,  darker,  and  much  more  markedly  granu- 
lar. In  shape  they  are  round,  oval,  and  lozenge-like. 
When  the  surface  colonies  are  viewed  under  a  low  power 
of  the  microscope  they  present  essentially  the  same  ap- 
pearance as  that  given  for  the  bacillus  of  typhoid  fever, 
viz.,  they  resemble  flattened  pellicles  of  glass-wall,  or 
patches  of  finely  ground  colorless  glass.  Colonies  of  this 
organism  on  gelatin  are  frequently  encountered  that 
cannot  be  distinguished  from  those  resulting  from  the 
growth  of  the  bacillus  of  typhoid  fever,  though,  as  a 
rule,  their  growth  is  a  little  more  luxuriant. 

In  stab  and  smear  cultures  on  gelatin  the  surface 
growth  is  flat,  dry,  and  blue-white  or  pearl  color. 
Limited  growth  occurs  along  the  track  of  the  needle  in 
the  depths  of  the  gelatin.  As  the  culture  becomes  older, 
the  gelatin  round  about  the  surface  growth  may  gradu- 
ally lose  its  transparency  and  become  cloudy,  often  quite 
opaque.  In  still  older  cultures  small  roots,  or  branch- 
like  projections  from  the  surface  growth  into  the  gelatin 
are  sometimes  seen  to  occur. 

It  does  not  cause  liquefaction  of  gelatin. 


BACTERIUM  COLT  COMMUNE.  325 

Its  growth  011  nutrient  agar-agar  and  on  blood-serum 
is  luxuriant  but  not  characteristic. 

In  bouillon  it  causes  diffuse  clouding  with  sedimenta- 
tion. In  some  bouillon  cultures  an  attempt  at  pellicle 
formation  on  the  surface  may  be  seen,  but  this  is  not 
always  the  case.  In  old  bouillon  cultures  the  reaction 
is  seen  to  have  become  alkaline,  and  a  decided  fa3cal  odor 
may  be  detected, 

It  produces  indol  in  bouillon  and  in  peptone  solu- 
tion. 

Its  growth  on  potato  is  rapid  and  voluminous,  appear- 
ing after  twenty-four  to  thirty-six  hours  in  the  incubator 
as  a  more  or  less  lobulated  layer  of  a  drab,  dark-cream, 
or  brownish-yellow  color. 

In  neutral  milk  containing  a  little  litmus  tincture  the 
blue  color  is  changed  to  red  after  from  eighteen  to 
twenty-four  hours  in  the  incubator,  and  in  addition,  the 
majority  of  cultures  cause  a  firm  coagulation  of  the 
casein  in  about  thirty-six  hours,  though  frequently  this 
takes  longer.  Very  rarely ,  the  litmus  may  indicate  the 
production  of  acid  and  no  coagulation  occur. 

In  media  containing  glucose  it  grows  rapidly  and 
causes  active  fermentation  with  liberation  of  carbonic 
acid  and  hydrogen.  If  cultivated  in  solid  media  to 
which  glucose  (2  per  cent.)  has  been  added,  the  gas- 
formation  is  recognized  by  the  appearance  of  numerous 
bubbles  along  and  about  the  points  of  growth.  If 
cultivated  in  fluid  media,  also  containing  glucose,  in  the 
fermentation-tube,  evidence  of  fermentation  is  given  by 
the  collection  of  gas  in  the  closed  arm  of  the  tube. 

On  lactose-litmus-agar-agar  its  colonies  are  pink  and 
the  color  of  the  surrounding  medium  is  changed  from 
blue  to  red. 

15 


326  BACTERIOLOGY. 

In  Dunham's  peptone  solution  it  produces  indol  in 
from  forty-eight  to  seventy-two  hours. 

It  stains  with  the  ordinary  aniline  dyes.  It  is  de- 
colorized when  treated  with  iodine  after  having  been 
stained  by  the  method  of  Gram. 

By  comparing  what  has  been  said  of  the  bacillus  typhi 
abdominalis  and  of  the  bacterium  coli  commune  it  will 
be  seen  that  while  they  simulate  each  other  in  certain 
respects  they  still  possess  individual  characteristics  by 
which  they  may  readily  be  differentiated.  The  most 
important  of  the  differential  points  are  : 

1.  Motility  of  the  bacillus  typhi  abdominalis  is  much 
more  conspicuous,  as  a  rule,  than  is  that  of  the  bacterium 
coli  commune. 

2.  On  gelatin  the  colonies  of  the  typhoid   bacillus 
develop  more  slowly  than  do  those  of  the  colon  bacillus. 

3.  On   potato  the  growth  of  the  typhoid  bacillus  is 
usually  invisible  (though  not  always),  while  that  of  the 
colon  bacillus  is  rapid,  luxuriant,  and  always  visible. 

4.  The  typhoid  bacillus  does  not  cause  coagulation  of 
milk  with  acid  reaction.     The  colon  bacillus  does  this 
in  from  thirty-six  to  forty-eight  hours  in  the  incubator. 

5.  The  typhoid  bacillus  never  causes  fermentation, 
with   liberation  of  gas,   in   media   containing   glucose, 
lactose,  or  saccharose.     The  colon  bacillus  is  conspicuous 
for  its  power  of  causing  fermentation  in  such  solutions. 

6.  In  nutrient  agar-agar  or  gelatin  containing  lactose 
and  litmus  tincture,  and  of  a  slightly  alkaline  reaction, 
the  color  of  the  colonies  of  typhoid  bacillus  is  blue,  and 
there  is  no  reddening  of  the  surrounding  medium,  while 
the  colonies  of  the  colon  bacillus  are  pink   and    the 
medium  round  about  them  becomes  red. 

7.  The  typhoid  bacillus  does  not  possess  the  property 


BACTERIUM  COLI  COMMUNE.  327 

of  producing  iudol  in  solutions  of  peptone ;  the  growth 
of  the  colon  bacillus  in  these  solutions  is  accompanied 
by  the  production  of  indol  in  from  forty-eight  to  seventy- 
two  hours  at  37°  to  38°  C. 

Animal  inoculations.  As  with  the  bacillus  of  typhoid 
fever,  the  results  of  inoculation  of  animals  with  cultures 
of  this  organism  cannot  be  safely  predicted.  According 
to  the  observations  of  Escherich,  Emmerich,  Weisser, 
and  others,  the  results  that  do  appear  are  in  most  in- 
stances to  be  attributed  to  the  toxic  rather  than  to  the 
infective  properties  of  the  culture  used. 

When  introduced  into  the  subcutaneous  tissues  of 
mice  it  has  no  effect,  while  similar  inoculations  of 
guinea-pigs  are  sometimes  (not  always)  followed  by 
abscess  formation  at  the  point  of  injury,  or  by  altera- 
tions very  similar  to  those  produced  by  intra- vascular 
inoculation,  viz.  :  death  in  less  than  twenty-four  hours, 
accompanied  by  redness  of  the  peritoneum  and  marked 
hypersemia  and  ecchymoses  of  the  small  intestine; 
together  with  swelling  of  Peyer's  patches.  The  caecum 
and  colon  may  remain  unchanged  or  present  enlarged 
follicles.  There  may  or  may  not  be  an  accumulation 
of  fluid  in  the  abdominal  cavity,  but  peritonitis  is  rarely 
present.  The  small  intestines  may  contain  bloody 
mucus. 

Intra-venous  inoculation  of  rabbits  may  be  followed 
by  similar  changes  with  often  the  occurrence  of  diar- 
rhoea before  death,  which  may,  in  the  acute  cases,  result 
in  from  three  to  forty  hours.  In  another  group  of 
cases  acute  fatal  intoxication  does  not  result,  and  the 
animal  lives  for  weeks  or  months,  dying  ultimately  of 
what  appears  to  be  the  effects  of  a  slow  or  chronic  form 
of  infection.  For  a  few  hours  after  inoculation  these 


328  BACTERIOLOGY. 

animals  present  no  marked  symptoms ;  exceptionally 
somnolence  and  diarrhoea  have  been  observed  at  this 
period,  indicating  acute  intoxication  from  which  the 
animal  has  recovered.  The  affection  is  unattended  by 
fever.  The  most  marked  symptom  is  loss  of  weight. 
This  is  usually  progressive  from  the  first  or  second  day 
after  inoculation,  with  slight  fluctuations  until  death. 

At  autopsy  the  animal  is  found  to  be  emaciated. 
The  subcutaneous  tissues  and  the  muscles  appear  pale 
and  dry.  The  serous  cavities,  particularly  the  peri- 
cardial,  may  contain  some  excess  of  serum.  The  viscera 
are  anaemic.  The  spleen  is  small,  thin,  and  pale.  Ex- 
ceptionally ulcers  and  ecchymoses  are  observed  in  the 
caecum,  but  generally  there  are  no  lesions  of  the  intes- 
tinal tract. 

The  most  striking  and  constant  lesions,  those  most 
characteristic  of  the  affection,  are  in  the  bile  and  in  the 
liver ;  the  quantity  of  bile  may  not  exceed  the  normal, 
but  in  other  cases  the  gall-bladder  may  be  abnormally 
distended  with  bile.  The  bile  is  nearly  colorless  or  has 
a  pale  yellowish  or  brownish  tint,  with  little  or  none  of 
a  greenish  color.  Its  consistence  is  much  less  viscid 
than  normal,  being  often  thin  and  watery.  It  usually 
contains  small,  opaque,  yellowish  particles  or  clumps 
which  can  be  seen  floating  in  it,  even  through  the  walls 
of  the  gall-bladder.  These  clumps  consist  microscopi- 
cally of  bile-stained,  apparently  necrotic,  epithelial 
cells ;  leucocytes  in  small  numbers ;  amorphous  masses 
of  bile  pigment,  and  bacteria  often  in  zoogloaa-like 
clumps.  Similar  material  is  found  in  the  larger  bile- 
ducts. 

The  liver  frequently  contains  opaque,  whitish  or 
yellowish-white  spots  and  streaks  of  irregular  size  and 


BACTERIUM  COLI  COMMUNE.  329 

shape,  which  give  a  peculiar  mottling  to  the  organ  when 
present  in  large  numbers.  These  areas  may  be  numer- 
ous, or  only  one  or  two  may  be  found.  In  size  they 
range  from  minute  points  to  areas  of  from  2  to  3  cm.  in 
extent. 

By  microscopic  examination  they  are  found  to  repre- 
sent places  where  the  liver  cells  have  undergone  necrosis 
accompanied  by  emigration  of  leucocytes,  and  the  cells 
about  them  are  in  a  condition  of  fatty  degeneration. 

In  sections  of  the  liver  masses  of  the  bacilli  may  be 
discovered  in  and  about  the  necrotic  foci  just  described. 

At  these  autopsies  the  colon  bacillus  is  not  found 
generally  distributed  through  the  body,  but  is  only  to 
be  detected  in  the  bile,  liver,  and  occasionally  in  the 
spleen.1 

1  Consult  paper  by  Blachstein  on  this  subject.    Johns  Hopkins  Hospital 
Bulletin,  July,  1891. 


CHAPTEE   XXII. 


The  spirillum  (comma  bacillus)  of  Asiatic  cholera— Its  morphological  and 
cultural  peculiarities— Pathogenic  properties— The  bacteriological  diagnosis 
of  Asiatic  cholera. 


AT  the  conference  held  in  Berlin  in  1884  for  the 
purpose  of  discussing  the  cholera  question,  it  was 
announced  by  Koch1  that  he  had  discovered  in  the 
intestinal  evacuations  of  individuals  suffering  from 
Asiatic  cholera  a  micro-organism  that  he  believed  to 
be  the  cause  of  the  malady.  The  importance  of  this 
statement  necessarily  attracted  widespread  attention  to 
the  subject,  and  as  one  of  the  results  there  existed,  for 
a  short  time  following,  some  skepticism  as  to  the  accu- 
racy of  Koch's  claim.  These  doubts  arose  as  a  result 
of  a  series  of  contributions  from  other  observers  who 
endeavored  to  prove  that  the  organism  found  by  Koch 
in  cholera  evacuations  was  one  that  is  common  to  other 
localities,  and  not  a  specific  accompaniment  of  this  dis- 
ease. It  was  not  very  long,  however,  before  it  was 
evident  that  the  objections  raised  by  the  opponents  of 
Koch  were  based  upon  untrustworthy  observations,  and 
that  by  reliable  methods  of  investigation  the  organism 
to  which  he  had  called  attention  could  be  easily  differ- 
entiated from  either  and  all  of  those  with  which  it  was 
claimed  to  be  identical. 

1  Verhandlungen  der  Conferenz  zur  Erorterung  der  Cholerafrage,  1884. 
Berlin. 


SPIRILLUM  OF  ASIATIC  CHOLERA.  331 

This  organism,  known  as  the  spirillum  of  Asiatic 
cholera,  and  as  Koch's  "  comma  bacillus/'  because  of  its 
morphology,  is  identified  by  the  following  peculiarities  : 

THE  MORPHOLOGICAL  AND  BIOLOGICAL  PECULIARITIES 
OF   THE   SPIRILLUM   OF   ASIATIC   CHOLERA. 

Morphology.  It  is  a  slightly  curved  rod  varying 
from  about  0.8  to  2.0  fj.  in  length  and  from  0.3  to  0.4/* 
in  thickness ;  that  is  to  say,  it  is  usually  from  about  one- 
half  to  two-thirds  the  length  of  the  tubercle  bacillus, 
but  is  thicker  and  plumper.  Its  curve  is  frequently  not 
more  marked  than  that  of  a  comma,  and,  indeed,*it  is 
often  almost  straight ;  at  times,  though,  the  curve  is 
much  more  pronounced,  and  may  even  describe  a  semi- 
circle. Occasionally  the  curve  may  be  double,  one  comma 
joining  another,  with  their  convexities  pointing  in  oppo- 
site directions,  so  that  a  figure  similar  to  the  letter  S  is 
produced.  In  cultures,  long  spiral  or  undulating  threads 
may  often  be  seen.  From  these  appearances  this  organ- 
ism cannot  be  considered  as  a  bacillus,  but  rather  as  an 
intermediate  type  between  the  bacilli  and  the  spirilla. 
Koch  thinks  it  not  improbable  that  the  short  comma 
forms  represent  segments  of  a  true  spirillum,  the  normal 
form  of  the  organism.  (Fig.  64.) 

It  does  not  form  spores,  and  we  have  no  reliable  evi- 
dence that  it  possesses  the  property  of  entering,  at  any 
time,  a  stage  when  its  powers  of  resistance  to  detrimental 
agencies  are  increased. 

It  is  a  flagellated  organism,  but  has  only  a  single 
flagellum  attached  to  one  of  its  ends. 

It  is  actively  motile,  especially  in  the  comma  stage, 
though  the  long  spiral  forms  also  possess  this  property. 


332  BACTERIOLOGY. 

Grouping.  As  found  in  the  slimy  flakes  in  the  intes- 
tinal discharges  from  cholera  patients,  Koch  likens  its 
mode  of  grouping  to  that  seen  in  a  school  of  small  fish 
when  swimming  up  stream,  i.  e.,  they  all  point  in  nearly 

FIG.  64. 


Spirillum  of  Asiatic  cholera.    Impression  cover-slip  from  a 
colony  thirty-four  hours  old. 

the  same  direction  and  lie  in  irregularly  parallel,  linear 
groups  that  are  formed  by  one  comma  being  located 
behind  the  other  without  being  attached  to  it. 

FIG.  65. 


Involution  forms  of  the  spirillum  of  Asiatic  cholera,  as  seen  in  old  cultures. 

On  cover-slip  preparations  made  from  cultures  in  the 
ordinary  way  there  is  nothing  characteristic  about  the 
grouping,  but  in  impression  cover-slips  made  from  young 
cultures  the  short  commas  will  nearly  always  be  seen  in 


SPIRILLUM  OF  ASIATIC  CHOLERA.  333 

small  groups  of  three  or  four,  lying  together  in  such  a 
way  as  to  have  their  long  axes  nearly  parallel  to  one 
another.  (See  Fig.  64.) 

In  old  cultures  in  which  development  has  ceased,  it 
undergoes  degenerative  changes,  and  the  characteristic 
comma  and  spiral  shapes  may  entirely  disappear,  their 
place  being  taken  by  irregular  involution  forms  that 
present  every  variety  of  outline.  (See  Fig.  65.)  In  this 
stage  they  take  on  the  staining  very  feebly,  and  often 
not  at  all. 

Cultural  peculiarities.  On  plates  of  nutrient  gelatin 
that  have  been  prepared  from  a  pure  culture  of  this 
organism  and  kept  at  a  temperature  of  from  20°  to  22° 
C.,  development  can  often  be  observed  after  as  short  a 
period  as  twelve  hours,  but  frequently  not  before  sixteen 
to  eighteen  hours.  This  is  especially  true  of  the  first 
or  "  original "  plate,  containing  the  largest  number  of 
colonies.  At  this  time  the  plate  will  present  to  the 
naked  eye  an  appearance  that  has  been  likened  to  a 
ground-glass  surface,  or  to  a  surface  that  has  been 
stippled  with  a  very  finely  pointed  needle,  or  one  upon 
which  very  fine  dust  has  been  sprinkled.  This  appear- 
ance is  due  to  the  presence  of  minute  colonies  closely 
packed  together  upon  the  surface  of  the  gelatin.  In  the 
depth  of  the  gelatin  can  also  be  seen,  closely  packed, 
small  points,  likewise  representing  growing  colonies. 
As  growth  progresses  liquefaction  occurs  around  the 
superficial  colonies,  and  in  consequence  this  plate  is  usu- 
ally entirely  liquid  after  from  twenty-four  to  thirty 
hours ;  the  developmental  phases  through  which  the 
colonies  pass  cannot,  therefore,  be  studied  upon  it. 

On  plates  2  and  3,  where  the  colonies  are  more  widely 
separated,  they  can  be  seen  after  twenty-four  to  thirty 

15* 


334 


BACTERIOLOGY. 


hours  as  small,  round,  or  oval,  white  or  cream-white 
points,  and  when  located  superficially  there  can  be  de- 
tected around  them  a  narrow  transparent  zone  of  lique- 
faction. As  growth  continues,  this  liquefaction  extends 
downward  rather  than  laterally,  and  the  colony  ulti- 
mately assumes  the  appearance  of  a  dense,  white  mass 
lying  at  the  bottom  of  a  sharply-cut  pit  or  funnel  con- 
taining transparent  fluid.  This  liquefaction  is  never 
very  widespread  nor  rapid,  and  rarely  extends  for  more 
than  one  millimetre  beyond  the  colony  proper.  On 
plates  containing  few  colonies  there  is  but  little  or  no 
tendency  for  them  to  become  confluent,  and,  as  a  rule, 
they  do  not  exceed  2  to  3  mm.  as  an  average  diameter. 

FIG.  66. 


c  d 

Developmental  stages  of  colonies  of  the  spirillum  of  Asiatic  cholera  at 
20°  to  22°  C.  on  gelatin.    X  about  75  diameters. 

a.  After  sixteen  to  eighteen  hours.  6.  After  twenty-four  to  twenty-six 
hours,  c.  After  thirty-eight  to  forty  hours,  d.  After  forty-eight  to  fifty 
hours,  e.  After  sixty-four  to  seventy  hours. 


When  examined  under  a  low  magnifying  lens  the 
very  young  colonies  (sixteen  to  eighteen  hours)  appear 
as  pale,  translucent,  granular  globules  of  a  very  delicate 
greenish  or  yellowish- green  color,  sharply  outlined  and 


SPIRILLUM  OF  ASIATIC  CHOLERA.  335 

not  perfectly  round.  (See  a,  Fig.  66.)  As  growth  pro- 
gresses, this  homogeneous  granular  appearance  is  re- 
placed by  an  irregular  lobulation,  and  ultimately  the 
sharply-cut  margin  of  the  colony  becomes  dentated  or 
scalloped.  (See  b  and  c,  Fig  66.)  After  forty-eight 
hours  (and  frequently  sooner),  liquefaction  of  the  gelatin 
has  taken  place  to  such  an  extent  that  the  appearance 
of  the  colony  is  entirely  altered.  Under  the  magnify- 
ing glass  the  colony  proper  is  now  seen  to  be  torn  and 
ragged  about  its  edges,  while  here  and  there  shreds  of 
the  colony  can  be  detected  scattered  through  the  liquid 
into  which  it  is  sinking.  These  shreds  evidently  repre- 
sent portions  of  the  colony  that  have  become  detached 
from  its  margin  as  it  gradually  sank  into  the  liquefied 
area. 

At  d,  in  Fig.  66,  will  be  seen  a  representation  of  the 
several  appearances  afforded  by  the  colonies  at  this 
stage.  At  the  end  of  the  second,  or  during  the  early 
part  of  the  third  day,  the  sinking  of  the  colonies  into 
the  liquefied  pits  resulting  from  their  growth  is  about 
complete,  and  under  a  low  lens  they  now  appear  as 
dense,  granular  masses,  surrounded  by  an  area  of  lique- 
faction through  which  can  be  seen  granular  prolonga- 
tions of  the  colony,  usually  extending  irregularly  between 
the  periphery  and  the  central  mass.  (See  e,  Fig  66.)  If 
the  periphery  be  examined,  it  will  be  seen  to  be  fringed 
with  delicate,  cilia-like  lines  that  radiate  from  it  in 
much  the  same  way  that  cilia  radiate  from  the  ends  of 
certain  columnar  epithelial  cells. 

These  are  the  more  marked  phases  through  which 
the  colonies  of  this  organism  pass  in  their  development 
on  gelatin  plates.  With  some  cultures  the  various  ap- 
pearances here  given  appear  more  quickly,  while  in 


336 


BACTERIOLOGY. 


cultures   from  other   sources   they   may  be    somewhat 
retarded. 

On  plates  of  nutrient  agar-agar  the  appearance  of  the 
colonies  is  not  characteristic.  They  appear  as  round  or 
oval  patches  of  growth  that  are  moist  and  tolerably 

PIG.  67. 


a  bed 

Stab  cultures  of  the  spirillum  of  Asiatic  cholera  in  gelatin, 

at  18°  to  20°  C. 

a.  After  twenty-four  hours.    &.  After  forty-eight  hours,    c.  After  seventy- 
two  hours,    d.  After  ninety-six  hours. 

transparent.  The  colonies  on  this  medium  at  37°  C. 
naturally  grow  to  a  larger  size  than  do  those  upon 
gelatin  at  22°  C. 

In  stab  cultures  in  gelatin  there  appears  at  the  top 
of  the  needle  track  after  thirty-six  to  forty-eight  hours 


SPIRILLUM  OF  ASIATIC  CHOLERA.  337 

at  22°  C.  a  small,  funnel-shaped  depression.  As  the 
growth  progresses,  liquefaction  will  be  seen  to  occur 
about  this  point.  In  the  centre  of  the  depression  can 
be  distinguished  a  small,  dense,  whitish  clump,  the 
colony  itself.  As  growth  continues  the  depression 
increases  in  extent  and  ultimately  assumes  an  appear- 
ance that  consists  in  the  apparent  sinking  of  the  liquefied 
portion  in  such  a  way  as  to  leave  a  perceptible  air-space 
between  the  top  of  the  liquid  and  the  surface  of  the  solid 
gelatin.  The  growth  now  appears  to  be  capped  by  a 
small  air-bubble.  The  impression  given  by  it  at  this 
stage  is  not  only  that  there  has  been  a  liquefaction,  but 
also  a  coincident  evaporation  of  the  fluid  from  the 
liquefied  area  and  a  constriction  of  the  superficial  open- 
ing of  the  funnel.  (See  a,  6,  c,  and  d,  Fig.  67.)  Lique- 
faction is  not  especially  active  along  the  deeper  portions 
of  the  track  made  by  the  needle,  though  in  stab  cultures 
in  gelatin  the  liquefaction  is  much  more  extensive  than 
that  usually  seen  around  colonies  on  plates.  It  spreads 
laterally  at  the  upper  portion,  and  after  about  a  week 
a  large  part  of  the  gelatin  in  the  tube  may  have  become 
fluid,  and  the  growth  will  have  lost  its  characteristic 
appearance. 

Stab-  and  smear-cultures  on  agar-agar  present  nothing 
characteristic.  They  are  usually  only  an  exaggeration 
of  the  appearance  afforded  by  the  single  colonies  on 
this  medium. 

Its  growth  in  bouillon  is  luxuriant,  causing  a  diffuse 
clouding  and  the  ultimate  production  of  a  delicate  film 
upon  the  surface. 

In  sterilized  milk  of  a  neutral  or  amphoteric  reac- 
tion at  a  temperature  of  36°-38°  C.  it  develops  actively, 
and  gradually  produces  an  acid  reaction  with  coagula- 


338  BACTERIOLOGY. 

tion  of  the  casein.  It  retains  its  vitality  under  these 
conditions  for  about  three  weeks  or  more.  The  blue 
color  of  milk  to  which  neutral  litmus  tincture  has  been 
added  is  changed  to  pink  after  thirty-six  or  forty-eight 
hours  at  body  temperature. 

Its  growth  in  peptone  solution,  either  that  of  Dun- 
ham (see  Special  Media)  or  the  one  preferred  by  Koch, 
viz.,  2  parts  Witte's  peptone,  1  part  sodium  chloride, 
and  100  parts  distilled  water,  is  accompanied  by  the 
production  of  both  indol  and  nitrites,  so  that  after 
eight  to  twelve  hours  in  the  incubator  at  37°  C.  the 
rose  color  characteristic  of  indol  appears  upon  the  addi- 
tion of  sulphuric  acid  alone.  (See  Indol  Reaction.) 

In  peptone  solution  to  which  rosolic  acid  has  been 
added  the  red  color  is  very  much  intensified  after  four 
or  five  days  at  37°  C. 

Its  growth  on  potato  of  a  slightly  acid  reaction  is 
seen  after  three  or  four  days  at  37°  C.  as  a  dull,  whit- 
ish, non-glistening  patch  at  and  about  the  site  of  inoc- 
ulation. It  is  not  elevated  above  the  surface  of  the 
potato,  and  can  only  be  distinctly  seen  when  held  to  the 
light  in  a  particular  position.  Growth  on  acid  potato 
occurs,  however,  only  at  or  near  the  body  temperature, 
owing  probably  to  the  acid  reaction,  which  is  sufficient 
to  prevent  development  at  a  lower  temperature,  but 
does  not  have  this  effect  when  the  temperature  is  more 
favorable. 

On  solidified  blood-serum  the  growth  is  usually  said 
to  be  accompanied  by  slow  liquefaction.  I  have  not 
succeeded  in  obtaining  this  result  on  Loeffler's  serum, 
nor  have  I  detected  anything  characteristic  about  its 
growth  on  this  medium. 

The  temperature  most  favorable  for  its  growth  is  be- 


SPIRILLUM  OF  ASIATIC  CHOLERA.  339 

tween  35°  and  38°  C.  It  grows,  but  more  slowly,  at 
17°  C.  Under  16°  C.  no  growth  is  visible. 

It  is  not  destroyed  by  freezing.  When  exposed  to 
65°  C.  its  vitality  is  destroyed  in  five  minutes. 

It  is  strictly  aerobic,  its  development  ceasing  if  the 
supply  of  oxygen  be  cut  off. 

It  does  not  grow  in  an  atmosphere  of  carbonic  acid, 
but  is  not  killed  by  a  temporary  exposure  to  this  gas. 
It  does  not  grow  in  acid  media,  but  flourishes  best  in 
media  of  neutral  or  slightly  alkaline  reaction.  It  is  so 
sensitive  to  the  action  of  acids  that  at  22°  C.,  its  devel- 
opment is  arrested  when  an  acid  reaction  equivalent  to 
0.066  to  0.08  per  cent,  hydrochloric  or  nitric  acid  is 
present  (Kitasato). 

In  cultures>  the  development  of  this  organism  reaches 
its  maximum  relatively  quickly,  then  remains  stationary 
for  a  short  period,  after  which  degeneration  begins.  The 
dying  comma  bacilli  become  altered  in  appearance  and 
assume  the  condition  known  as  "  involution  forms." 
(See  Fig.  65.)  When  in  this  state  they  take  up  color- 
ing reagents  very  faintly  or  not  at  all,  and  may  lose  en- 
tirely their  characteristic  shape. 

When  present  with  other  bacteria,  under  conditions 
favorable  to  growth,  the  comma  bacillus  at  first  grows 
much  more  rapidly  than  do  the  others  ;  in  twenty-four 
hours  it  will  often  so  outnumber  the  other  organisms 
present  that  microscopic  examination  would  lead  one 
to  take  the  material  under  consideration  to  be  a  pure 
culture  of  this  organism.  This,  however,  does  not  last 
longer  than  two  or  three  days  ;  they  then  begin  to  die, 
and  the  other  organisms  gain  the  ascendency.  This  fact 
has  been  taken  advantage  of  by  Schottelius1  in  the  fol- 

i  Deutsche  med.  Wochenschrift,  1885,  No.  14. 


340  BACTERIOLOGY. 

lowing  method  devised  by  him  for  the  bacteriological 
examination  of  dejections  from  cholera  patients  : 

In  dejections  that  are  not  examined  immediately  after 
being  passed  it  is  often  difficult,  because  of  the  large 
number  of  other  bacteria  that  may  be  present,  to  detect 
with  certainty  the  cholera  organism  by  microscopic  ex- 
amination. It  is  advantageous  in  these  cases  to  mix  the 
dejections  with  about  double  their  volume  of  slightly 
alkaline  beef  tea,  and  allow  them  to  stand  for  about 
twelve  hours  at  a  temperature  of  between  30°  and  40°  C. 
There  appears  at  the  end  of  this  time,  especially  upon 
the  surface  of  the  fluid,  a  conspicuous  increase  in  the 
number  of  comma  bacilli,  and  cover-slip  preparations 
made  from  the  upper  layers  of  the  fluid  will  reveal  an 
almost  pure  culture  of  this  organism. 

It  is  not  improbable  that  a  similar  process  occurs  in 
the  intestines  of  those  suffering  from  Asiatic  cholera, 
viz.,  a  rapid  multiplication  of  the  comma  bacilli  that 
have  gained  access  to  the  intestines  takes  place,  but  lasts 
for  only  a  short  time,  when  the  comma  bacilli  begin  to 
disappear,  and  after  a  few  days  their  place  is  taken  by 
other  organisms. 

In  connection  with  his  experiments  upon  the  poison 
produced  by  the  cholera  organism,  Pfeiffer1  states  that 
in  very  young  cultures,  grown  under  the  access  of  oxy- 
gen, there  is  present  a  poisonous  body  that  possesses 
intense  toxic  properties.  This  primary  cholera- poison 
stands  in  very  close  relation  to  the  material  composing 
the  bodies  of  the  bacteria  themselves,  and  is  probably  an 
integral  constituent  of  them,  for  the  vitality  of  the  cholera 
spirilla  can  be  destroyed  by  means  of  chloroform  and 

1  Zeitschrift  f.  Hygiene  u.  Infectionskrankheiten,  Bd.  xi.,  p.  393. 


SPIRILLUM  OF  ASIATIC  CHOLERA.  341 

thymol,  and  by  drying,  without,  apparently,  any  altera- 
tion of  this  poisonous  body.  Absolute  alcohol,  concen- 
trated solutions  of  neutral  salts  and  a  temperature  of 
100°  C.,  decompose  this  substance,  leaving  behind 
secondary  poisons  which  possess  a  similar  physiologi- 
cal activity,  but  only  when  given  in  from  ten  to  twenty 
times  the  dose  necessary  to  produce  the  same  effects  with 
the  primary  poison. 

Other  members  of  the  vibrio  family  also,  namely,  the 
vibrio  Metchnikovi  and  that  of  Finkler  and  Prior  (see 
description  of  these  species),  contain,  according  to  Pfeiffer, 
closely  related  poisons. 

Experiments  upon  animals.  As  a  result  of  experi- 
ments for  the  purpose  of  determining  if  the  disease  can 
be  produced  in  any  of  the  lower  animals,  it  is  found  that 
white  mice,  monkeys,  cats,  dogs,  poultry,  and  many 
other  animals  are  not  susceptible  to  infection  by  the 
methods  usually  employed  in  inoculation  experiments. 
When  animals  are  fed  on  pure  cultures  of  the  comma 
bacillus  no  effect  is  produced,  and  the  organisms  cannot 
be  obtained  from  the  stomach  or  intestines;  they  are 
destroyed  in  the  stomach  and  do  not  reach  the  intes- 
tines ;  they  are  not  demonstrable  in  the  faBces  of  these 
animals.  Intra-vascular  injections  of  pure  culture  into 
rabbits  are  followed  by  a  temporary  illness,  from  which 
the  animals  usually  recover  in  from  two  to  three  days ; 
intra-peritoneal  injections  into  white  mice  are,  as  a  rule, 
followed  by  death  in  from  twenty-four  to  forty-eight 
hours ;  the  conditions  in  both  instances  most  probably 
resulting  from  the  toxic  activities  of  the  poisonous  prod- 
ucts of  growth  of  the  organisms  that  are  present  in  the 
culture  employed.  None  of  the  lower  animals  have  ever 


342  BACTERIOLOGY. 

been  known  to   suffer   from  Asiatic   cholera   spontan- 
eously. 

The  failure  to  induce  cholera  in  animals  by  feeding, 
or  by  injection  of  cultures  into  the  stomach,  was  shown 
by  Nicati  and  Rietsch l  to  be  due  to  the  destructive  action 
of  the  acid  gastric  juice  on  the  bacilli.  They  showed 
that  if  cultures  of  this  organism  were  introduced  into 
the  alimentary  tract  of  certain  animals  in  such  a  manner 
that  they  would  not  be  subjected  to  the  influence  of  the 
gastric  juice,  a  condition  pathologically  closely  simu- 
lating cholera  as  it  occurs  in  man,  could  be  produced. 
For  this  purpose  the  common  bile  duct  was  ligated,  after 
which  the  cultures  were  injected  directly  into  the  duod- 
enum. Such  interference  with  the  flow  of  bile  lessens 
intestinal  peristalsis,  and  thus  permits  the  development 
of  the  bacilli  at  the  point  at  which  they  are  deposited, 
that  is,  the  portion  of  the  intestine  having  an  alkaline 
reaction  and  beyond  the  influence  of  the  acid  stomach- 
juice. 

By  this  method  Nicati  and  Rietsch,  Van  Ermeugem,2 
Koch,3  and  others  were  enabled  to  produce  in  the 
animals  upon  which  they  operated  a  condition  that 
was,  if  not  identical,  at  all  events  very  similar  patho- 
logically to  that  seen  in  the  intestine  of  subjects  dead 
of  the  disease. 

At  a  subsequent  conference  held  in  Berlin  in  1885 
Koch4  described  the  following  method  by  means  of  which 
he  had  been  able  to  obtain  a  relatively  high  degree  of 
constancy  in  all  his  efforts  to  produce  cholera  in  lower 

1  Archiv.  de  Phys.  norm.  et.  path.  1885,  xvii.,  3e  ser.,  t.  vi.    Compt.-rend., 
xcix.  p.  928.    Rev.  de  Hygiene,  1885.    Rev.  de  Med.,  1885,  v. 

2  "  Recherches  sur  le  Microbe  du  Cholera  Asiatique."  Paris- Bruxelles,  1885. 
Bull,  de  1'Acad.  roy.  de  M6d.  de  Belgique,  3e  s6r.,  xviii. 

3  Loc.  cit.  4  LOC.  cit.,  1885. 


SPIE1LL  UM  OF  ASIATIC  CHOLERA.  343 

animals :  Bearing  in  mind  the  point  made  by  Nicati  and 
Rietsch  as  to  the  effect  produced  by  the  acid  reaction  of 
the  gastric  juice,  this  reaction  was  first  to  be  neutralized 
by  injecting  through  a  soft  catheter  passed  down  the 
oesophagus  into  the  stomach,  5  c.c.  of  a  5  per  cent,  solu- 
tion of  sodium  carbonate.  Ten  or  fifteen  minutes  later 
this  was  to  be  followed  by  the  injection  into  the  stomach 
(also  through  a  soft  catheter)  of  10  c.c.  of  a  bouillon  cul- 
ture of  the  cholera  spirillum.  For  the  purpose  of  arrest- 
ing peristalsis  and  permitting  the  organism  to  remain  in 
the  stomach  and  upper  part  of  the  duodenum  for  as  long 
a  time  as  possible,  the  animal  was  to  receive,  immedi- 
ately following  the  injection  of  the  culture,  an  intra- 
peritoneal  injection,  by  means  of  a  hypodermic  syringe, 
of  1  c.c.  of  tincture  of  opium  for  each  200  grammes  of 
its  body  weight.  Shortly  after  this  last  injection  a  deep 
narcosis  sets  in  and  lasts  from  a  half  to  one  hour,  after 
which  the  animal  is  again  as  lively  as  ever.  Of  35 
guinea-pigs  inoculated  in  this  way  by  Koch,  30  died  of 
a  condition  that  was,  in  general,  very  similar  to  that 
seen  in  Asiatic  cholera. 

The  condition  of  these  animals  before  death  is  de- 
scribed as  follows:  Twenty-four  hours  after  the  opera- 
tion the  animal  appears  sick;  there  is  a  loss  of  appetite, 
and  the  animal  remains  quiet  in  its  cage.  On  the  fol- 
lowing day  a  paralytic  condition  of  the  hind  extremities 
appears,  which,  as  the  day  goes  on,  becomes  more  pro- 
nounced ;  the  animal  lies  quite  flat  upon  its  abdomen  or 
on  its  side,  with  its  legs  extended  ;  respiration  is  weak 
and  prolonged,  and  the  pulsations  of  the  heart  are  hardly 
perceptible ;  the  head  and  extremities  are  cold,  and  the 
body  temperature  is  frequently  subnormal. 


344  BACTERIOLOGY. 

The  animal  usually  dies  after  remaining  in  this  con- 
dition for  a  few  hours. 

At  autopsy  the  small  intestine  is  found  to  be  deeply 
injected  and  filled  with  a  flocculent,  colorless  fluid. 
The  stomach  and  intestines  do  not  contain  solid  masses, 
but  fluid ;  when  diarrhoea  does  not  occur,  firm  scybala 
may  be  expected  in  the  rectum.  Both  by  microscopic 
examination  and  by  culture  methods,  comma  bacilli  are 
found  to  be  present  in  the  small  intestine  in  practically 
pure  culture. 

More  recently  Pfeiffer1  has  determined  that  essentially 
similar  constitutional  effects  may  be  produced  in  guinea- 
pigs  by  the  intra-peritoneal  injection  of  relatively  large 
numbers  of  this  organism.  His  plan  is  to  scrape  from 
the  surface  of  a  fresh  culture  on  agar-agar  as  much  of 
the  growth  as  can  be  held  upon  a  moderate-sized  wire 
loop.  This  is  then  finely  divided  in  1  c.c.  of  bouillon 
and,  by  means  of  a  hypodermic  syringe,  is  injected 
directly  into  the  peritoneal  cavity.  When  virulent  cul- 
tures have  been  used  this  is  quickly  followed  by  a  fall 
in  the  temperature  of  the  animal ;  this  is  gradual  and 
continuous  until  death  ensues,  which  is  usually  in  from 
eighteen  to  twenty-four  hours  after  the  operation,  though 
exceptionally  cases  do  occur  in  which  the  animal  recov- 
ers, even  after  having  exhibited  marked  symptoms  of 
most  profound  toxaemia. 

In  pursuance  of  his  studies  upon  this  disease,  Pfeiffer2 
has  demonstrated  that  it  is  possible  to  render  an  animal 
tolerant  or  immune  to  the  poisonous  properties  of  this 
organism  by  repeated  injections  of  non-fatal  doses  of 
dead  cultures  (cultures  that  have  been  killed  by  the 

1  Zeitschrift  fur  Hygiene,  Bd.  xi.  and  xiv. 

2  Zeit.  fur  Hyg.  u.  Infections  Krankheiten,  Bd.  ix.,  Heft.  i. 


SPIRILLUM  OF  ASIATIC  CHOLERA.  345 

vapor  of  chloroform  or  by  heat).  He  also  demonstrated 
that  the  serum  of  animals  so  immunified  possesses  a 
specific  germicidal  action  toward  the  cholera  spirillum, 
i.  e.,  if  into  the  peritoneal  cavity  of  an  animal  immuni- 
fied against  Asiatic  cholera,  living  cholera  spirilla  be 
introduced,  they  will  all  be  destroyed  (disintegrated) 
within  a  relatively  short  time.  Furthermore,  if  the 
serum  of  an  animal  immunified  against  cholera  be 
injected  into  the  peritoneal  cavity  of  a  similar  animal 
not  so  immunified,  and  immediately  afterward  living 
cholera  spirilla  be  introduced,  a  similar  disintegration 
and  destruction  of  the  bacteria  will  also  result.  He 
shows  that  a  more  or  less  definite  relation  exists  between 
the  amount  of  serum  and  the  number  of  organisms  in- 
troduced. Such  a  destruction  of  the  comma  bacillus  by 
the  serum  of  an  immunified  animal  does  not  occur  out- 
side the  animal  body,  that  is,  cannot  be  demonstrated 
in  a  test  tube.  The  specificity  of  this  reaction  is  sug- 
gested by  Pfeiifer  as  a  means  of  differentiating  the  cholera 
spirillum  from  other  suspicious  species,  for  no  such  dis- 
integration of  bacterial  cells  is  observed  if  species  other 
than  the  cholera  spirillum  be  introduced  into  the  peri- 
toneal cavity  of  animals  immunified  against  Asiatic 
cholera. 

General  considerations.-  In  all  cases  of  Asiatic  cholera, 
and  only  in  this  disease,  the  organism  just  described  can 
be  detected  in  the  intestinal  evacuations.  The  more 
acute  the  case  and  the  more  promptly  the  examination 
is  made  after  the  evacuations  have  been  passed  from  the 
patient,  the  less  will  be  the  difficulty  experienced  in 
detecting  the  organism. 

In  some  cases  it  can  be  detected  in  the  vomited  mat- 


346  BACTERIOLOGY. 

ters,  though  by  no  means  so  constantly  as  in  the  intes- 
tinal contents. 

As  a  rule,  bacteriological  examination  fails  to  reveal 
the  presence  of  the  organisms  in  the  blood  and  internal 
organs  in  this  disease,  though  Nicati  and  Rietsch  claim 
to  have  obtained  them  from  the  common  bile-duct  in 
rapidly  fatal  cases,  and  in  two  out  of  five  cases  they 
were  present  in  the  gall-bladder.  Doyen  and  Rasst- 
schewsky1  found  them  in  the  liver  in  pure  culture, 
and  Tizzoni  and  Cattani2  in  both  the  blood  and  the  gall- 
bladder. 

The  cholera  spirillum  is  a  facultative  parasite ;  that 
is  to  say,  it  apparently  finds  in  certain  portions  of  the 
world,  particularly  in  those  countries  in  which  Asiatic 
cholera  is  endemic,  conditions  that  are  not  entirely  un- 
favorable to  its  development  outside  of  the  body.  This 
has  been  found  to  be  the  fact  not  only  by  Koch,  who 
detected  the  presence  of  the  organism  in  the  water-tanks 
in  India,  but  by  many  other  observers  who  have  suc- 
ceeded in  demonstrating  its  growth  under  conditions  not 
embraced  in  the  ordinary  methods  that  are  employed 
for  the  cultivation  of  bacteria. 

The  results  of  experiments  having  for  their  object 
the  determination  of  the  length  of  time  during  which 
this  organism  may  retain  its  vitality  in  water  are  con- 
spicuous for  their  irregularity.  In  the  transactions  of 
the  congress  in  Berlin,  for  the  discussion  of  the  cholera 
question,  it  is  stated,  in  connection  with  this  point,  that 
the  experiments  made  with  tank- water  in  India  some- 
times resulted  in  demonstrating  the  multiplication  of 

1  Reference  to  Vratch,  1885,  in  Allg.  Med.  Central.  Zeitung,  Berlin. 

2  Centralblatt  f.  die  med.  Wissenschaften,  1886,  No.  43. 


SPIRILLUM  OF  ASIATIC  CHOLERA.  347 

the  organisms  introduced  into  it,  while  in  other  cases 
they  died  very  quickly. 

On  February  8,  1884,  comma  bacilli  were  found  in 
the  tank  at  Saheb-Began,  in  Calcutta,  and  it  was  pos- 
sible to  demonstrate  them  in  a  living  condition  up  to 
February  23d. 

Koch  states  that  in  ordinary  spring-water  or  well- 
water  the  organisms  retained  their  vitality  for  thirty 
days,  whereas  in  the  canal-water  (sewage)  of  Berlin  they 
died  after  six  or  seven  days ;  but  if  this  latter  were  mixed 
with  faecal  matters,  the  organisms  retained  their  vitality 
for  but  twenty-seven  hours ;  and  in  the  undiluted  con- 
tents of  cesspools  it  is  impossible  to  demonstrate  them 
after  twelve  hours.  In  the  experiments  of  Nicati  and 
Rietsch  they  retained  their  vitality  in  sterilized  distilled 
water  for  twenty  days  ;  in  Marseilles  canal-water  (sew- 
age), for  thirty- eight  days ;  in  sea- water,  sixty-four 
days ;  in  harbor- water,  eighty-one  days  ;  and  in  bilge- 
water,  thirty-two  days. 

In  the  experiments  of  Hochstetter,  on  the  other  hand, 
they  died  in  distilled  water  in  less  than  twenty-four 
hours  in  five  of  seven  experiments  ;  in  one  of  the  two 
remaining  experiments  they  were  alive  after  a  day,  and 
in  the  other  after  seven  days. 

In  one  experiment  with  the  domestic  water  supply  of 
Berlin  the  organism  retained  its  vitality  for  267  days; 
in  another  for  382  days,  notwithstanding  the  fact  that 
many  other  organisms  were  present  at  the  same  time. 
There  is  no  single  ground  upon  which  these  variations 
can  be  explained,  for  they  depend  apparently  upon  a 
number  of  factors  which  may  act  singly  or  together. 
For  example,  in  general  it  may  be  said  that  the  higher 
the  temperature  of  the  water  in  which  these  organisms 


348  BACTERIOLOGY. 

are  present,  up  to  20°  C.,  the  longer  do  they  retain  their 
vitality  ;  the  purer  the  water,  that  is,  the  poorer  in 
organic  matters,  the  more  quickly  do  the  organisms  die, 
whereas  the  richer  it  is  in  organic  matter  the  longer  do 
they  retain  their  vitality. 

Still  another  point  that  must  be  considered  in  this 
connection  is  the  antagonistic  influences  under  which 
they  find  themselves  when  placed  in  water  containing 
large  numbers  of  organisms  that  are,  so  to  speak,  at 
home  in  water — the  so-called  normal  water  bacteria. 

The  effect  of  light  upon  growing  bacteria  must  not 
be  lost  sight  of,  for  it  has  been  shown  that  a  surprisingly 
large  number  of  these  organisms  are  robbed  of  their 
vitality  by  a  relatively  short  exposure  to  the  rays  of  the 
sun,  and  it  is,  therefore,  not  unlikely  that  the  non- 
observance  of  this  fact  may  be,  in  part  at  least,  account- 
able for  some  of  the  discrepancies  that  appear  in  the 
results  of  these  experiments. 

In  his  studies  upon  the  behavior  of  pathogenic  and 
other  micro-organisms  in  the  soil,  Carl  Frankel1  found 
that  the  cholera  spirillum  was  not  markedly  susceptible 
to  those  deleterious  influences  that  cause  the  death  of  a 
number  of  other  pathogenic  organisms.  During  the 
months  of  August,  September,  and  October,  cultures 
of  the  comma  bacillus  that  had  been  buried  in  the 
ground  at  a  depth  of  three  metres  retained  their 
vitality ;  on  the  other  hand,  in  other  mouths,  particu- 
larly from  April  to  July,  they  lost  their  vitality  when 
buried  to  the  depth  of  only  two  metres.  At  a  depth 
of  one  and  a  half  metres  vitality  was  not  destroyed, 
and  there  was  a  regular  development  in  cultures  so 
placed. 

i  Zeitschrift  f.  Hygiene,  Bd.  ii.  p.  521. 


SPIRILLUM  OF  ASIATIC  CHOLERA.  349 

As  a  result  of  experiments  performed  in  the  Imperial 
Health  Bureau,  at  Berlin,  it  was  found  that  the  bodies 
of  guinea-pigs  that  had  died  of  cholera  induced  by 
Koch's  method  of  inoculation  contained  no  living  cholera 
spirilla  when  exhumed  after  having  been  buried  for 
nineteen  days  in  wooden  boxes,  or  for  twelve  days  in 
zinc  boxes.  In  a  few  that  had  been  buried  in  moist 
earth,  without  having  been  encased  in  boxes,  when  ex- 
humed after  two  or  three  months,  the  results  of  exami- 
nations for  cholera  spirilla  were  likewise  negative. 

Kitasato,1  in  his  experiments  with  the  cholera  organ- 
ism, found  that  when  mixed  with  the  intestinal  evacua- 
tions of  human  beings  under  ordinary  conditions,  they 
lost  their  vitality  in  from  a  day  and  a  half  to  three 
days.  If  the  evacuations  were  sterilized  before  the  cul- 
tures were  mixed  with  them,  the  organisms  retained  their 
vitality  up  to  from  twenty  to  twenty-five  days.  He  was 
unable  to  come  to  any  definite  conclusion  as  to  the  cause 
of  these  phenomena. 

It  was  demonstrated  by  Hesse2  and  by  Celli3  that 
many  substances  commonly  employed  as  food  stuffs  offer 
a  favorable  nidus  upon  which  the  cholera  organism  may 
develop.  In  his  experiments  upon  its  behavior  in  milk, 
Kitasato4  found  that  at  a  temperature  of  36°  C.  the 
cholera  spirillum  developed  very  rapidly  during  the  first 
three  or  four  hours,  and  outnumbered  the  other  organ- 
isms commonly  found  in  milk.  They  then  diminished 
in  number  from  hour  to  hour  as  the  acidity  of  the  milk 
increased,  until  finally  their  vitality  was  lost;  at  the 

1  Zeitschrift  fur  Hygiene,  Bd.  v.  p.  487. 

2  Ibid.,  Bd.  v.  p.  527. 

3  Bolletino  della  R.  Accad.  Med.  di  Roma,  1888. 
*  Zeitschrift  f.  Hygiene,  Bd.  v.  p.  491. 

16 


350  BACTERIOLOGY. 

same  time  the  common  saprophytic  bacteria  increased  in 
number.  Relatively  the  same  process  occurs  at  a  lower 
temperature,  from  22°  to  25°  C.,  but  the  process  is 
slower,  the  maximum  development  of  the  cholera  organ- 
isms being  reached  at  about  the  fifteenth  hour,  after 
which  time  they  were  overgrown  by  the  ordinary  sapro- 
phytes present. 

From  this  it  would  seem  that  the  vitality  of  the 
cholera  spirillum  in  milk  depends  largely  upon  the 
reaction  :  the  more  quickly  the  milk  becomes  sour  the 
more  quickly  does  the  organism  become  inert,  while 
the  longer  the  milk  retains  its  neutral,  or  only  very 
slightly  acid  reaction,  the  longer  do  the  cholera  organ- 
isms that  may  be  present  in  it  retain  their  power  of 
multiplication. 

According  to  Laser,1  the  cholera  organism  retains  its 
vitality  in  butter  for  about  seven  days ;  it  is  therefore 
possible  for  the  disease  to  be  contracted  by  the  use  of 
butter  that  has  in  any  way  been  in  contact  with  cholera 
material. 

In  regard  to  the  antagonism  between  the  cholera 
spirillum  and  other  organisms  with  which  it  may  come 
in  contact,  the  experiments  of  Kitasato2  led  him  to 
conclude  that  no  organism  has  been  found  which,  when 
growing  in  the  same  culture  medium  with  it,  possessed 
the  power  of  depriving  it  of  its  vitality  within  a  short 
time.  On  the  other  hand,  the  experiments  showed  that 
there  were  quite  a  number  of  other  organisms  the  devel- 
opment of  which  was  checked,  and  in  some  cases  their 
vitality  was  completely  destroyed,  when  growing  in  the 
same  medium  with  the  cholera  spirillum. 

1  Zeitschrift  fur  Hygiene,  Bd.  x.  p.  513. 

2  Ibid.,  Bd.  vi.  p.  1. 


SPIRILLUM  OF  ASIATIC  CHOLERA.  351 

From  this  it  would  appear  that  the  disappearance  of 
the  cholera  spirillum  from  mixed  cultures  and  from  the 
evacuations  in  so  short  a  time  as  has  been  mentioned,  is 
due  more  to  unfavorable  nutritive  condition  than  to  the 
direct  action  of  the  other  organisms  present. 

When  completely  dried,  according  to  Koch's  experi- 
ments, the  cholera  spirillum  does  not  retain  its  vitality 
for  longer  than  twenty-four  hours,  but  by  others  its 
vitality  is  said  to  be  destroyed  by  an  absolute  drying  of 
three  hours.  In  the  moist  conditions,  as  in  artificial 
cultures,  vitality  may  be  retained  for  many  months, 
though  repeated  observations  lead  us  to  believe  that, 
under  these  circumstances,  the  virulence  is  diminished. 
According  to  Kitasato,1  they  retain  their  vitality  when 
smeared  upon  thin  glass  cover-slips  and  kept  in  the 
moist  chamber  for  from  85  to  100  days,  and  for  as 
long  as  200  days  when  deposited  upon  bits  of  silk 
thread. 

In  the  course  of  his  studies  upon  the  destiny  of 
pathogenic  micro-organisms  in  the  dead  body,  Von 
Esmarch2  found  that,  when  the  cadaver  of  a  guinea- 
pig  dead  from  the  introduction  of  cholera  organisms 
into  the  stomach,  was  immersed  in  water  and  decom- 
position allowed  to  set  in,  after  eleven  days,  when  de- 
composition was  far  advanced,  it  was  impossible  to  find 
any  living  cholera  spirilla  by  the  ordinary  plate  methods. 

A  similar  experiment  resulted  in  their  disappearance 
after  five  days.  In  another  experiment,  in  which  de- 
composition was  allowed  to  go  on  without  the  animal 
being  immersed  in  water,  none  could  be  detected  after 
the  fifth  day. 

1  Zeitschrift  fur  Hygiene,  Bd.  v.  p.  134; 

2  Ibid.,  Bd.  vii.  p.  1. 


352  BACTERIOLOGY. 

Carl  Frankel1  has  shown  that  an  atmosphere  of 
carbonic  acid  is  directly  inhibitory  to  the  development 
of  the  cholera  spirillum,  and  Percy  Frankland2  states 
that  in  an  atmosphere  of  this  gas  it  dies  in  about  eight 
days.  In  an  atmosphere  of  carbon  monoxide  its  vitality 
is  lost  in  nine  days,  and  in  general  the  same  may  be 
said  for  it  when  under  the  influence  of  an  atmosphere 
of  nitrous  oxide  gas. 

From  what  has  been  said  we  see  that  the  spirillum 
of  Asiatic  cholera,  while  possessing  the  power  of  pro- 
ducing in  human  beings  one  of  the  most  rapidly  fatal 
forms  of  disease  with  which  we  are  acquainted,  is  still 
one  of  the  least  resistant  of  the  pathogenic  organisms 
known  to  us.  Under  conditions  most  favorable  to  its 
growth,  its  development  is  self-limited ;  it  is  conspicu- 
ously susceptible  to  acids,  alkalies,  other  chemical  disin- 
fectants, and  heat ;  but  when  partly  dried  upon  clothing, 
food,  or  other  objects,  it  may  retain  its  vitality  for  a  rela- 
tively long  period  of  time,  and  it  is  more  than  probable 
that  it  is  in  this  way  that  the  disease  is  often  carried 
from  points  in  which  it  is  epidemic  or  endemic,  into 
localities  that  are  free  from  the  disease. 


THE   DIAGNOSIS   OF   ASIATIC   CHOLERA    BY   BACTERIO- 
LOGICAL  METHODS. 

Because  of  the  manifold  channels  that  are  open  for 
the  dissemination  of  this  disease  it  is  of  the  utmost 
importance  that  its  true  nature  should  be  recognized  as 
quickly  as  possible,  for  with  every  moment  of  delay  in 
its  recognition  opportunities  for  its  spread  are  multi- 


Zeitschrift  fur  Hygiene,  Bd.  v.  p.  332.  2  jbid.,  Bd.  vi.  p.  13. 


DIAGNOSIS  OF  ASIATIC  CHOLERA.  353 

plying.  It  is  essential,  therefore,  when  employing  bac- 
teriological means  in  making  the  diagnosis,  to  bear  in 
mind  those  biological  and  morphological  features  of  the 
organism  that  appear  most  quickly  under  artificial 
methods  of  cultivation,  and  which,  at  the  same  time, 
may  be  considered  as  characteristic  of  it,  viz.,  its  peculiar 
morphology  and  grouping ;  the  much  greater  rapidity 
of  its  growth  over  that  of  other  bacteria  with  which  it 
may  be  associated  ;  the  characteristic  appearance  of  its 
colonies  on  gelatin-plates  and  of  its  growth  in  stab 
cultures  in  gelatin  ;  its  property  of  producing  indol  and 
coincidently  nitrites  in  from  six  to  eight  hours  in  pep- 
tone solution  at  37°  to  38°  C.;  and  its  power  of  causing 
the  death  of  guinea-pigs  in  from  sixteen  to  twenty-four 
hours  when  introduced  into  the  peritoneal  cavity,  death 
being  preceded  by  symptoms  of  extreme  toxaemia,  char- 
acterized by  prostration  and  gradual  and  continuous  fall 
in  the  temperature  of  the  animal's  body. 

In  a  publication  recently  made  by  Koch1  he  called 
attention  to  a  plan  of  procedure  that  is  employed  in 
this  work  in  the  Institute  for  Infectious  Diseases  at 
Berlin.  In  this  scheme  the  points  that  have  been 
enumerated  are  taken  into  account,  and  by  its  employ- 
ment the  diagnosis  can  be  established  in  the  majority 
of  cases  of  Asiatic  cholera  in  from  eighteen  to  twenty- 
two  hours.  In  general  the  steps  to  be  taken  and  points 
to  be  borne  in  mind  are  as  follows.  The  material 
should  be  examined  as  early  as  possible  after  it  has 
been  passed. 

I.  Microscopic*  examination.  From  one  of  the  small 
slimy  particles  that  will  be  seen  in  the  semi-fluid  evac- 

i  Zeitschrift  fiir  Hygiene,  1893,  Bd.  xiv. 


354  BACTERIOLOGY. 

uations,  prepare  a  cover-slip  preparation  in  the  ordinary 
way  and  stain  it.  If,  upon  microscopic  examination, 
only  curved  rods,  or  curved  rods  greatly  in  excess  of 
all  other  forms,  are  present,  the  diagnosis  of  Asiatic 
cholera  is  more  than  likely  correct ;  and  particularly  is 
this  true  if  these  organisms  are  arranged  in  irregular 
linear  groups  with  the  long  axes  of  all  the  rods  point- 
ing in  nearly  the  same  direction,  that  is  to  say,  some- 
what as  minnows  arrange  themselves  when  swimming  in 
schools  up  stream.  (Koch.) 

In  1886  Weisserand  Frank1  expressed  their  opinion 
upon  the  value  of  microscopic  examination  in  these  cases 
in  the  following  terms  : 

(a)  In  the  majority  of  cases  microscopic  examination 
is  sufficient  for  the  detection  of  the  presence  of  the 
comma  bacillus  in  the  intestinal  evacuations  of  cholera 
patients. 

(6)  Even  in  the  most  acute  cases,  running  a  very 
rapid  course,  the  comma  bacillus  can  always  be  found 
in  the  evacuations. 

(c)  In  general,-  the  number  of  cholera  spirilla  present 
is  greater  the  earlier  death  occurs ;  when  death  is  post- 
poned, and  the  disease  continues  for  a  longer  period, 
their  number  is  diminished. 

(d)  Should  the  patient  not  die  of  cholera,  but  from 
some  other  disease,  such  as  typhoid  fever,  that  may  be 
engrafted   upon  it,  the  comma   bacilli   may  disappear 
entirely  from  the  intestines. 

II.  With  another  slimy  flake  prepare  a  set  of  gelatin 
plates.  Place  them  at  a  temperature  of  from  20°  to 
22°  C.,  and  at  sixteen,  twenty-two,  and  thirty-six  hours 

i  Zeitschrift  fur  Hygiene,  Bd.  i.,  p.  397. 


DIAGNOSIS  OF  ASIATIC  CHOLEEA.  355 

observe  the  appearance  of  the  colonies.  Usually  at 
about  twenty-two  hours  the  colonies  of  this  organism 
can  easily  be  identified  by  one  familiar  with  them. 

III.  With  another  slimy  flake  start  a  culture  in  a  tube 
of  peptone  solution — either  the  solution  of  Dunham  or, 
as  Koch  proposes,  a  solution  of  double  the  strength  of 
that  of  Dunham  (Witte's  peptone  is  to  be  used,  as  it 
gives  the  best  and  most  constant  results).  Place  this  at 
37°  to  38°  C.,  and  at  the  end  of  from  six  to  eight  hours 
prepare  cover-slips  from  the  upper  layers  (without  shak- 
ing) and  examine  them  microscopically ;  if  comma 
bacilli  were  present  in  the  original  material,  and  are 
capable  of  multiplication,  they  will  be  found  in  this 
locality  in  almost  pure  culture.  After  doing  this  pre- 
pare a  second  peptone  culture  from  the  upper  layers  of 
the  one  just  examined,  also  a  set  of  gelatin  plates,  and 
with  what  remains  make  the  test  for  indol  by  the  addi- 
tion of  ten  drops  of  concentrated  sulphuric  acid  for  each 
ten  cubic  centimetres  of  fluid  contained  in  the  tube.  If 
comma  bacilli  are  growing  in  the  tube  the  rose  color 
characteristic  of  the  presence  of  indol  should  appear. 

By  following  this  plan  "a  bacteriologist  who  is 
familiar  with  the  morphological  and  biological  pecu- 
liarities of  this  organism  should  make  a  more  than 
probable  diagnosis  at  once  by  microscopic  examination 
alone,  and  a  positive  diagnosis  in  from  twenty  to,  at  the 
most,  twenty-four  hours  after  beginning  the  examina- 
tion." (Koch.) 

There  are  certain  doubtful  cases  in  which  the  organ- 
isms are  present  in  the  intestinal  canal  in  very  small 
numbers,  and  microscopic  examination  is  not,  therefore, 
of  so  much  assistance.  In  these  cases  plates  of  agar- 


356  BACTERIOLOGY. 

agar,  of  gelatin,  and  cultures  in  the  peptone  solution 
should  be  made. 

The  plates  of  agar- agar  should  not  be  prepared  in  the 
usual  way,  but  the  agar-agar  should  be  poured  into 
Petri  dishes  and  allowed  to  solidify,  after  which  one  of 
the  slimy  particles  may  be  smeared  over  its  surface. 
The  comma  bacillus,  being  markedly  aerobic,  develops 
very  much  more  readily  when  its  colonies  are  located 
upon  the  surface  than  when  they  are  in  the  depths  of 
the  medium.  A  point  to  which  Koch  calls  attention,  in 
connection  with  this  step  in  the  manipulation,  is  the 
necessity  for  having  the  surface  of  the  agar-agar  free 
from  the  water  that  is  squeezed  from  it  when  it  solidi- 
fies, as  the  presence  of  the  water  interferes  with  the  de- 
velopment of  the  colonies  as  isolated  points  and  causes 
them  to  become  confluent.  To  obviate  this  he  recom- 
mends that  the  agar-agar  be  poured  into  the  plates  and 
the  water  allowed  to  separate  from  the  surface  at  the 
temperature  of  the  incubator  before  they  are  used.  It  is 
wise,  therefore,  when  one  is  liable  to  be  called  on  for 
such  work  as  this  to  keep  a  number  of  sterilized  plates 
of  agar-agar  in  the  incubator  ready  for  use,  just  as  ster- 
ilized tubes  of  media  are  always  ready  and  at  hand.  The 
advantage  of  using  the  agar  plates  is  the  higher  temper- 
ature at  which  they  can  be  kept,  and  consequently  a  more 
favorable  condition  for  the  development  of  the  colonies. 
As  soon  as  isolated  colonies  appear  they  should  be  ex- 
amined microscopically  for  the  presence  of  organisms 
having  the  morphology  of  the  one  for  which  we  are  seek- 
ing, and  as  soon  as  such  is  detected  gelatin  plates  and 
cultures  in  peptone  solution  (for  the  indol  reaction) 
should  be  made.  The  peptone  cultures  started  from 
the  original  material  should  be  examined  microscopi- 


DIAGNOSIS  OF  ASIATIC  CHOLERA.  357 

cally  from  hour  to  hour  after  the  sixth  hour  that  they 
have  been  in  the  incubator.  The  material  taken  for 
examination  should  always  come  from  near  the  surface 
of  the  fluid,  and  care  should  be  taken  not  to  shake  the 
tube.  As  soon  as  comma  bacilli  are  detected  in  anything 
like  considerable  numbers  in  the  upper  layers  of  the 
fluid,  agar-agar  plates  and  fresh  peptone  cultures  should 
be  made  from  them.  The  colonies  will  develop  on  the 
agar-agar  plates  at  37°  C.  in  from  ten  to  twelve  hours 
to  a  size  sufficient  for  recognition  by  microscopic  ex- 
amination, and  from  this  examination  an  opinion  can 
usually  be  given.  This  opinion  should  always  be  con- 
trolled by  cultures  in  the  peptone  solution  made  from 
each  of  several  single  colonies,  and  finally  the  test 
for  the  presence  or  absence  of  indol  in  these  cultures. 
In  all  doubtful  cases  in  which  only  a  few  curved 
bacilli  are  present,  or  in  which  irregularities  in  either 
the  rate  or  mode  of  their  development  occurs,  pure  cul- 
tures should  be  obtained  by  the  agar-agar-plate  method 
and  by  the  method  of  cultivation  in  peptone  solution,  as 
soon  as  possible,  and  their  virulence  tested  upon  animals. 
For  this  purpose  cultures  upon  agar-agar  from  single 
colonies  must  be  made.  From  the  surface  of  one  of  such 
cultures  a  good  sized  wire-loopful  should  be  scraped 
and  this  broken  up  in  about  one  cubic  centimetre  of 
bouillon,  and  the  suspension  thus  made  injected  by 
means  of  a  hypodermic  syringe  directly  into  the  per- 
itoneal cavity  of  a  guinea-pig  of  about  350  to  400 
grammes  weight.  For  larger  animals  more  material 
should  be  used.  If  the  material  injected  is  from  &  fresh 
culture  of  the  cholera  organism  toxic  symptoms  at  once 
begin  to  appear ;  these  have  their  most  pronounced  ex- 
pression in  the  lowering  of  temperature,  and  if  one  fol- 

16* 


358  BACTERIOLOGY. 

lows  this  decline  in  temperature  from  time  to  time  with 
the  thermometer  it  will  be  seen  to  be  gradual  and  con- 
tinuous from  the  time  of  injection  to  the  death  of  the 
animal  (Pfeiffer1),  which  occurs  in  from  eighteen  to 
twenty-four  hours  after  the  operation. 

In  general,  this  is  the  procedure  employed  in  the 
Institute  for  Infectious  Diseases,  at  Berlin,  tinder 
Koch's  direction. 

*  Loc.  cit. 


CHAPTER  XXIII. 


Organisms  of  interest,  historically  and  otherwise,  that  have  been  confounded 
with  the  spirillum  of  Asiatic  cholera— Their  peculiarities  and  differential 
features— The  vibrio  proteus,  or  bacillus  of  Finkler  and  Prior— The  spirillum 
tyrogenum,  or  cheese  spirillum  of  Deneke— The  spirillum  of  Miller— The  vibrio 
Metchnikovi. 


VIBRIO   PROTEUS  (FINKLER-PRIOR   BACILLUS). 

Finkler  and  Prior  were  the  first  to  contest  experi- 
mentally the  significance  of  the  presence  of  Koch's 
comma  bacillus  in  Asiatic  cholera,  claiming  to  have 
found  it  in  the  dejections  of  individuals  suffering  from 
other  maladies,  particularly  cholera  nostras.  The  mor- 
phological and  biological  differences  between  the  organ- 
ism that  Finkler  and  Prior  had  discovered  and  those  of 
the  comma  bacillus  described  by  Koch  are,  however,  so 
pronounced  as  to  warrant  the  opinion  that  the  confusion 
had  arisen  through  imperfect  and  untrustworthy  methods 
of  experimentation.  At  a  somewhat  later  period  Finkler 
and  Prior  retracted  their  claims  of  identity  for  the  two 
organisms,  and  held  that  the  bacterium  with  which  they 
were  dealing  was  peculiar  to  cholera  nostras — an  opinion 
which,  in  the  light  of  subsequent  work,  was  also  proved 
to  be  without  foundation  in  fact. 

The  characteristics  of  the  spirillum  of  Finkler  and 
Prior  are  as  follows  : 

* 

MORPHOLOGY. — It  is  thicker  and  longer  than  the 
spirillum  of  Asiatic  cholera  ;  it  is  often  thicker  at  the 


360  BACTERIOLOGY. 

middle  than  at  the  poles  ;  it  forms,  like  the  "  comma 
bacillus,"  screw-like,  twisted  threads  (Fig.  68). 

It  is  supplied  with  a  single  flagellum  at  one  of  its 
ends,  and  is,  therefore,  motile. 

It,  like  the  comma  bacillus,  readily  undergoes 
degenerative  changes  under  conditions  unfavorable  to 
growth  and  presents  the  variety  of  shapes  grouped  under 
the  head  "involution  forms."  According  to  Buchner 
this  is  especially  the  case  when  the  medium  in  which 
they  are  growing  contains  glucose  (5  per  cent.)  or  gly- 
cerin (2  per  cent.). 

FIG.  68. 


Vibrio  proteus,  Finkler-Prior  bacillus,  from  culture  on  agar-agar  twenty- 
four  hours  old. 


CULTURAL  PECULIARITIES. — On  gelatin  plates  the 
development  of  its  colonies  is  far  more  rapid,  and 
liquefaction  far  more  extensive,  than  in  the  case  of  the 
cholera  spirillum.  After  twenty-two  to  twenty-four 
hours  in  this  medium  at  20°  to  22°  C.  the  average  size 
of  the  colonies  is  about  double  that  of  the  comma  bacillus. 
The  colonies  are  darker  and  denser  and  do  not  present 
under  the  low  lens  the  same  degree  of  granulation  and 
subsequent  lobulation,  and  they  do  not  become  serrated 
or  scalloped  around  the  margin  as  is  the  case  with  Koch's 
organism.  After  twenty-two  to  twenty-four  hours  they 
are  usually  nearly  round,  regularly  granular,  and  more 


VIBRIO  PROTEUS.  361 

or  less  sharply  defined.  (See  Fig.  69,  a.)  At  times 
they  may  show  indefinite  markings  or  creases,  somewhat 
suggestive  of  lobulations.  After  forty-eight  hours  on 
gelatin  they  usually  range  from  one  to  three  millimetres 
(some  even  larger)  in  diameter,  and  will  appear  as 
sharply  cut,  saucer-shaped  pits  of  liquefaction,  in  the 
most  dependent  portion  of  which  lies  a  dense,  irregular 
mass,  the  colony  proper.  Under  low  magnifying  power 
they  present  at  this  stage  an  appearance  similar  to  that 
shown  in  Fig.  69,  b,  the  central  dense  mass  representing 

FIG.  69. 


am 


Colonies  of  the  Fiiikler-Prior  bacillus  on  gelatin,    x  about  75  diameters, 
a.  After  twenty-two  hours  at  20°  to  22°  C.    6.  After  forty-eight  hours  at  20° 

to  22°  C. 

the  colony  and  the  irregular  ragged  lines  surrounding  it 
being  shreds  that  have  become  torn  away  as  it  sank  into 
the  liquid  caused  by  its  growth.  The  zone  surrounding 
it,  extending  to  the  periphery,  is  somewhat  cloudy,  and 
is  simply  liquefied  gelatin.  There  is  a  marked  tendency 
for  the  liquefaction  to  spread  laterally  and  for  the 
colonies  to  run  together,  so  that,  even  on  plates  con- 
taining few  colonies,  in  sixty  to  seventy-two  hours  at 


362 


BACTERIOLOGY. 


•from  20°  to  22°  C.,  the  entire  gelatin  is  usually  con- 
verted into  a  yellowish-white  fluid.  Under  these  con- 
ditions its  growth  is  accompanied  by  a  marked  aromatic 
odor,  impossible  to  describe ;  this  is  especially  the  case 
when  the  liquefaction  is  far  advanced. 


FIG.  70. 


abed 

Stab  culture  of  the  Finkler-Prior  bacillus  in  gelatin  at  18°  to  20°  C. 

a.  After  twenty-four  hours.    &.  After  forty -eight  hours,    c.  After  seventy-two 

hours,    d.  After  ninety-six  hours. 

In  stab  cultures  in  gelatin  at  the  room  temperature, 
liquefaction  is  noticed  about  the  upper  part  of  the 
needle-track  iu  twenty- four  hours.  This  condition 
gradually  increases,  and  at  the  end  of  two  or  three  days 
the  entire  upper  portion  of  the  gelatin  has  become  con- 


VIBRIO  PROTEUS.  363 

verted  into  a  cloudy  fluid,  whereas  at  the  lower  part  of 
the  canal  the  liquefaction  progresses  less  rapidly  but  is 
still  much  more  marked  than  that  seen  as  a  result  of 
the  growth  of  Koch's  spirillum.  Indeed,  under  these 
circumstances  there  is  no  similarity  whatever  between 
the  growth  of  the  two  organisms  (see  a,  6,  c,  d,  Fig  70, 
and  compare  these  with  corresponding  cuts  in  Fig.  67). 

It  is  customary  to  see,  scattered  through  the  cloudy 
liquefied  gelatin,  ragged,  more  or  less  dense  masses, 
fragments  of  the  colony  proper. 

On  nutrient  agar-agar  there  is  nothing  particularly 
characteristic  about  its  growth,  appearing  only  as  a 
moist,  grayish  or  yellowish -gray  deposit. 

On  potato,  after  forty-eight  to  seventy-two  hours,  there 
appears  a  pale,  yellowish-gray  deposit;  this  is  moist, 
glazed,  and  marked  by  lobulations,  and  is  surrounded 
by  an  irregular,  colorless  zone  of  growth  that  is  much 
less  moist  than  that  forming  the  central  area.  It  grows 
well  on  potato  at  the  ordinary  temperature  of  the  room. 

It  causes  liquefaction  of  solidified  blood-serum  and  of 
coagulated  egg  albumin. 

In  milk  to  which  neutral  litmus  tincture  has  been 
added  the  blue  color  takes  on  a  pink  tinge  in  from  two 
to  three  days  at  37°  to  38°  C. 

It  does  not  form  indol  nor  does  it  cause  fermentation 
of  glucose. 

In  peptone  solution  containing  rosolic  acid,  the  color 
is  somewhat  deepened  after  four  or  five  days  at  37°  C. 

EXPERIMENTS  UPON  ANIMALS. — By  ordinary  methods 
of  inoculation  this  organism  is  without  pathogenic  pro- 
perties. Injections,  subcutaneous  and  intra-vascular 
and  directly  into  the  stomach,  give  negative  results. 
When  introduced  into  the  stomach  of  guinea-pigs  by  the 


364  BACTERIOLOGY. 

method  employed  by  Koch  in  his  cholera  experiments, 
Finkler  and  Prior  had  3  out  of  10  animals,  and  Koch  5 
out  of  15  animals  so  treated  to  die. 

The  claim  of  Finkler  and  Prior  that  this  organism 
was  related  etiologically  to  cholera  nostras  has  been 
shown  by  subsequent  work  to  have  been  unjustifiable. 

In  1885,  1886,  and  1887,  Franck1  examined  seven 
cases  that  clinically  presented  the  condition  of  cholera 
nostras ;  in  none  of  these  seven  cases  was  the  organism 
of  Finkler  and  Prior,  which  they  claimed  to  be  the 
cause  of  the  disease,  found.  In  all  cases  the  results  of 
bacteriological  examination,  in  so  far  as  the  constant 
presence  of  an  organism  that  might  stand  in  causal  re- 
lation to  the  disease  was  concerned,  were  negative.  Only 
the  ordinary  intestinal  bacteria  were  found. 


SPIEILLUM  TYROGENUM  (CHEESE  SPIRILLUM  OF 
DENEKE). 

Another  spiral  form,  likewise  forming  short,  comma- 
shaped  segments  in  the  course  of  its  growth  (Fig.  71),  is 
that  found  by  Deneke  in  old  cheese.  It  is  a  little  smaller 
than  Koch's  spirillum.  It  is  motile  and  has  but  a  single 
flagellum,  attached  to  one  of  its  ends.  It  liquefies  gelatin 
more  rapidly  than  does  Koch's  organism.  It  possesses  no 
characteristic  grouping,  as  can  be  seen  in  impression  cover- 
slips  of  its  colonies.  It  does  not  form  spores.  On  gelatin 
plates  its  colonies  develop  very  rapidly  as  saucer-shaped 
depressions  ;  after  twenty-four  hours  they  vary  from  1  to 
4  mm.  in  transverse  diameter.  To  the  naked  eye  they 
are  almost  transparent,  and  are  usually  marked  by  a 

i  Zeitschrift  f.  Hygiene,  Bd.  iv.  p.  207. 


SPIRILLUM  TYROGENUM.  365 

denser  centre  and  peripheral  zone,  the  space  between 
being  quite  clear.  They  are  not  regularly  round  in  all 
cases.  A  peculiar  aromatic  odor  accompanies  their 
growth  on  gelatin.  Under  a  low  magnifying  power 


\ 


Deneke's  cheese  spirillum,  spirillum  tyrogenum.    From  agar-agar  culture 
twenty-four  hours  old. 

the  smallest  colonies  are  irregularly  round  in  outline, 
their  borders  being  often  rough  and  broken,  and  the 
body  of  the  colony  is  frequently  marked  by  creases  or 
ridges  that  give  to  it  a  lobulated  appearance.  The 


Colony  oi  spirillum  tyrogenum  on  gelatin,  twenty-four  hours  old. 

larger  colonies  under  the  same  lens  appear  as  granular 
patches,  a  little  denser  at  the  periphery  and  centre  than 
at  the  intermediate  portions.  The  periphery  gradually 
fades  away  and  no  distinct  circumference  can  be  made 
out.  (See  Fig.  72.)  The  colonies  of  an  intermediate 


366 


BAC1ER10LOGY. 


size,  about  which  liquefaction  is  just  beginning  to  be  ap- 
parent, show  a  dense  granular  centre,  the  colony  itself, 
and  round  about  it  a  delicate,  granular  developmental 
zone. 

In   stab   cultures   in   gelatin    liquefaction   is   rapid, 
causing  at  the  end  of  twenty-four  hours  a  cup-shaped 

FIG.  73. 


\ 

a  b  c  d 

Stab  culture  of  Deneke's  cheese  spirillum  in  gelatin,  at  18°  to  20°  C. 

a.  After  twenty-four  hours.    &.  After  forty-eight  hours,    c.  After  seventy-two 

hours,    d.  After  ninety-six  hours. 

depression  at  the  top  of  the  needle-track,  the  superficial 
area  of  which  is  about  half  that  of  the  gelatin  in  the 
tube.  (Fig.  73,  a.)  The  liquefying  process  spreads 
laterally  and  at  the  end  of  forty-eight  hours  the  whole 
upper  portion  of  the  gelatin  may  have  become  liquid. 


SPIRILLUM  TYROGENUM.  367 

(Fig.  73,  6.)  This  process  continues  along  the  track  of 
the  needle  and  after  seventy-two  and  ninety- six  hours 
the  appearances  shown  in  Fig.  73,  c  and  d,  will  be  pro- 
duced. 

There  is  nothing  particularly  characteristic  about  its 
growth  upon  agar-agar. 

On  potato  there  appears  a  moist,  glazed,  yellowish, 
and,  at  points,  brownish-yellow  growth  that  is  sur- 
rounded by  a  drier,  colorless  zone.  It  is  not  lobulated. 

In  milk  containing  neutral  litmus  tincture  a  pink 
color  appears  after  two  to  three  days  at  37°  C. ;  after 
four  days  the  milk  is  almost  decolorized  and  there  is 
beginning  to  appear  coagulation  of  the  casein  with  a 
layer  of  clear  whey  above  it.  During  the  subsequent 
twenty-four  hours  there  is  complete  separation  of  the 
contents  of  the  tube  into  clot  and  whey. 

In  Dunham's  peptone  solution  it  does  not  form  indol 
and  the  reaction  for  this  body  does  not  appear  with 
either  sulphuric  acid  alone  or  plus  sodium  nitrite. 

It  causes  liquefaction  of  both  coagulated  blood-serum 
and  egg  albumin. 

There  is  no  pellicle  formed  as  a  result  of  its  growth 
in  bouillon. 

It  does  not  produce  fermentation  of  glucose. 

In  rosolic-acid-peptone  solution  its  growth  causes  the 
red  color  to  become  deepened  after  four  or  five  days  at 
37°  C. 

By  Koch's  method  of  introducing  cultures  into  the 
stomach  of  guinea-pigs  this  organism  produced  the  death 
of  three  out  of  fifteen  animals  experimented  upon — the 
deaths  resulting,  most  probably,  more  from  the  toxic 
action  of  the  products  of  growth  that  were  introduced 


368  BACTERIOLOGY. 

with  the  organisms  than  to  any  pathogenic  powers  pos- 
sessed by  the  organism  itself. 


Another  spirillum  that  has  been  likened  to  that  of 
Koch  is  the  one  obtained  by  Miller  from  a  carious 
tooth.  It  has  so  many  characteristics  in  common  with 
the  organism  of  Finkler  and  Prior  that  Miller  was 
inclined  to  consider  them  identical.  In  morphology 
they  are  indistinguishable.  (See  Fig.  74.)  It  grows 

FIG.  74. 


Spirillum  of  Miller.    From  agar-agar  culture  twenty-four  hours  old. 

rapidly,  and,  like  the  spirillum  of  Finkler  and  Prior, 
causes  rapid  liquefaction  of  gelatin  with  the  coincident 
production  of  a  peculiar  aromatic  odor. 

The  colonies  on  gelatin  plates  appear  after  twenty- 
four  hours  as  small,  transparent  pits  of  liquefaction  in 
the  centre  of  which  can  be  seen  a  minute  white  point,  the 
colony  itself.  Under  a  low  lens  the  largest  of  these 
points  are  uniformly  granular  and  regularly  round,  and 
as  a  rule  are  surrounded  by  a  peripheral  zone  that  is 
a  little  darker  than  the  central  portion  of  the  colony. 
The  circumference  is  delicately  fringed  by  short,  cilia- 
like  prolongations  of  growth  which  are  not  as  a  rule 
straight,  but  are  twisted  in  all  directions  and  can  only 


MILLERS  SPIRILL UM.  369 

be  detected  upon  very  careful  examination.  (See  a,  Fig. 
75.)  When  located  deep  in  the  gelatin  the  colonies 
are  round,  sharply  circumscribed,  of  a  pale-yellowish  or 
greenish-yellow  color,  and  marked  by  very  delicate 
irregular  lines  or  ridges.  After  forty-eight  hours  the 
plate  containing  many  colonies  is  entirely  liquefied, 
while  that  containing  only  a  few  shows  the  presence  of 
round,  sharply  cut,  shallow  pits  of  liquefaction  that 
measure  from  two  to  ten  mm.  in  diameter.  They  are 
a  little  denser  at  the  centre  than  at  the  periphery,  and 
the  dense  centre  is  not  sharply  circumscribed,  but  fades 
off  into  what  has  the  appearance  of  a  delicate  film. 
(See  6,  Fig.  75.)  As  the  colonies  become  older  they 

FIG.  75. 


6 
Colonies  of  Miller's  spirillum  on  gelatin,  at  20°  to  22°  C.  X  about  fifty-seven 

diameters. 

a.  Colony  just  beneath  the  surface  of  the  gelatin.    6,  Colony  on  the  surface 
of  the  gelatin. 

are  sometimes  marked  by  irregular  radii  extending  from 
periphery  to  centre  like  the  spokes  of  a  wheel. 

In  stab  cultures  in  gelatin  it  rapidly  produces  lique- 
faction, both  at  the  surface  and  along  the  needle-track, 
and  in  most  respects  gives  rise  to  a  condition  very  like 
that  resulting  from  the  growth  of  Finkler  and  Prior's 
spirillum,  though  differing  from  it  in  certain  details. 
(See  a,  b,  c,  d,  Fig.  76.) 

On  agar-agar  nothing  of  special  interest  appears  as  a 
result  of  its  development. 


370 


BACTERIOLOGY. 


On  potato  its  growth  is  very  like  that  of  the  cholera 
spirillum,  viz.,  it  appears  at  37°  C.  as  a  dry,  white 
patch  that  lies  quite  flat  upon  the  surface  and  can  often 
only  be  seen  when  the  tube  is  held  to  the  light  in  a 
special  way. 

FIG.  76. 


a  bed 

Stab  culture  of  Miller's  spirillum  in  gelatin,  at  18°  to  20°  C. 

a.  After  twenty-four  hours.   6.  After  forty-eight  hours,    c.  After  seventy-two 

hours,    d.  After  ninety-six  hours. 

Its  growth  in  bouillon  is  not  characteristic.  It  does 
not  form  a  pellicle. 

It  causes  liquefaction  of  both  coagulated  blood-serum 
and  egg  albumin. 

It  does  not  produce  indol. 

It  does  not  cause  fermentation  of  glucose. 

It  is  non-motile. 


VIBRIO  METCHNIKOVI.  371 

In  milk  containing  blue  litmus  tincture  it  causes 
almost  complete  decolorization  in  from  three  to  four 
days  at  37°  C.,  with  coincident  coagulation  of  the 
casein  and  the  formation  of  a  layer  of  clear  whey 
about  it. 

It  causes  the  red  color  of  rosolic-acid- pep  tone  solution 
to  become  somewhat  intensified  after  four  or  five  hours 
at  37°  C. 

Of  twenty-one  animals  treated  with  this  organism  by 
Koch's  method  of  inoculation  only  four  died. 

VIBRIO   METCHNIKOVI. 

The  spirillum  that  simulates  very  closely  the  comma 
bacillus  of  cholera  in  its  morphological  and  cultural 
peculiarities,  but  which  is  still  easily  distinguished  from 
it,  is  that  described  by  Gamaleia  under  the  name  of 

FIG.  77. 


Vibrio  Metchnikovi  from  agar-agar  culture,  twenty-four  hours  old. 

vibrio  Metchnikovi.  It  was  found  post-mortem  in  a 
number  of  fowls  that  had  died  in  the  poultry  market  of 
Odessa,  and  the  experiments  of  the  discoverer  demon- 
strate that  it  is  related  etiologically  to  the  gastro-enter- 
itis  with  which  the  chickens  had  been  suffering. 

In  morphology  it  is  seen  as  short,  curved  rods  and  as 


372  BACTERIOLOGY. 

longer,  spiral-like  filaments.  It  is  usually  thicker  than 
Koch's  spirillum  and  is  at  times  much  longer,  while 
again  it  is  seen  to  be  shorter.  It  is  usually  more  dis- 
tinctly curved  than  the  "comma  bacillus."  (Fig.  77.) 

It  is  supplied  with  a  single  flagellum  at  one  of  its 
extremities  and  is,  therefore,  motile. 

It  does  not  form  spores. 

It  is  aerobic. 

Its  growth  upon  gelatin  plates  is  usually  characterized, 
according  to  Pfeiffer,  by  the  appearance  of  two  kinds 
of  liquefying  colonies,  one  strikingly  like  those  of  the 
Finkler-Prior  organism,  the  other  very  similar  to  those 
produced  by  Koch's  comma  bacillus,  though  in  both 
cases  the  liquefaction  resulting  from  the  growth  of  this 
organism  is  more  energetic  than  that  common  to  the 
spirillum  of  Asiatic  cholera.  After  from  twenty-four 
to  thirty  hours  the  medium-sized  colonies,  when  exam- 
ined under  a  low  power  of  the  microscope,  show  a  yel- 
lowish-brown, ragged  central  mass  surrounded  by  a  zone 
of  liquefaction  that  is  marked  by  a  border  of  delicate 
radii.  (Fig.  78.) 

FIG.  78. 


Colony  of  vibrio  Metchnikmn.  in  gelatin,  after  thirty  hours  at  20°  to  22°  C. 
X  about  75  diameters. 


In  gelatin  stab  cultures  the  growth  has  much  the  same 
general  appearance  as  that  of  the  cholera  spirillum,  but 
is  very  much  exaggerated  in  degree.  The  liquefaction  is 
far  more  rapid  and  the  characteristic  appearance  of  the 
growth  is  lost  in  from  three  to  four  days.  (See  a,  6, 


VIBRIO  METCHNIKOVI. 


373 


c,  d,  Fig.  79.)  Development  and  liquefaction  along 
the  deeper  parts  of  the  needle-track  are  much  more  pro- 
nounced than  is  the  case  with  the  "comma  bacillus." 


FIG.  79. 


a  6  c  d 

Stab  culture  of  vibrio  Metchnikovi  in  gelatin,  at  18°  to  20°  C. 
a.  After  twenty -four  hours,     b.  After  forty-eight  hours,     c.  After  seventy- 
two  hours,    d.  After  ninety-six  hours. 

Its  growth  on  agar-agar  is  rapid,  and  after  twenty- 
four  to  forty-eight  hours  there  appears  a  grayish  deposit 
having  a  tendency  to  take  on  a  yellowish  tone. 

On  potato  at  37°  C.  its  growth  is  seen  as  a  moist, 
coffee-colored  patch  surrounded  by  a  much  paler  zone. 
The  whole  growth  is  so  smooth  and  glistening  that  it 
has  almost  the  appearance  of  being  varnished, 

17 


374  BACTERIOLOGY. 

In  bouillon  it  quickly  causes  opacity,  with  the  ulti- 
mate production  of  a  delicate  pellicle  upon  the  surface. 

It  causes  liquefaction  of  blood-serum,  the  liquefied 
area  being  covered  by  a  dense,  wrinkled  pellicle. 

When  grown  in  peptone  solution  it  produces  indol 
and  coincidently  nitrites,  so  that  the  rose-colored  reac- 
tion characteristic  of  indol  is  obtained  by  the  addition 
of  sulphuric  acid  alone.  The  production  of  indol  by 
this  organism  is  usually  greater  than  that  common  to 
the  comma  bacillus  under  the  same  circumstances. 

In  milk  it  causes  an  acid  reaction  with  cogulation 
of  the  casein.  The  coagulated  casein  collects  at  the 
bottom  of  the  tube  in  irregular  masses,  above  which  is 
a  layer  of  clear  whey.  If  blue  litmus  has  been  added 
to  the  milk  the  color  is  changed  to  pink  by  the  end  of 
twenty-four  to  thirty  hours,  and  after  forty-eight  hours 
decolorization  and  coagulation  occurs.  The  clots  of 
casein  are  not  re-dissolved.  After  about  a  week  the 
acidity  of  the  milk  is  at  a  maximum,  and  the  organ- 
isms quickly  die. 

It  causes  the  red  color  of  the  rosolic-acid -peptone  solu- 
tion to  become  very  much  deeper  after  four  or  five  days 
at  37°  C. 

It  does  not  cause  fermentation  of  glucose  with  pro- 
duction of  gas. 

It  is  killed  in  five  minutes  by  a  temperature  of  50° 
C.  (Sternberg.) 

It  is  pathogenic  for  chickens,  pigeons,  and  guinea- 
pigs.  Rabbits  and  mice  are  affected  only  by  very  large 
doses. 

Chickens  affected  with  the  choleraic  gastro  enteritis, 
of  which  this  organism  is  the  cause,  are  usually  seen 
sitting  quietly  about  with  ruffled  feathers.  They  are 


VIBRIO  METCHNIKOVI.  375 

afflicted  with  diarrhoea,  but  do  not  have  any  elevation 
of  temperature.  A  hypersemia  of  the  entire  gastro- 
intestinal tract  is  seen  at  autopsy.  The  other  internal 
organs  do  not,  as  a  rule,  present  anything  abnormal  to 
the  naked  eye.  The  intestinal  canal  contains  yellowish 
fluid  with  which  blood  may  be  mixed.  In  adult 
chickens  the  spirilla  are  not  found  in  the  blood,  but  in 
young  ones  they  are  usually  present  in  small  numbers. 
By  subcutaneous  inoculation,  pigeons  succumb  to  the 
pathogenic  activities  of  this  organism  in  from  eight  to 
twelve  hours.  At  autopsy  pretty  much  the  same  con- 
dition is  seen  as  was  described  for  chickens,  except  that 
large  numbers  of  the  spirilla  are  usually  present  in 
the  blood.  Guinea-pigs  usually  die  in  from  twenty 
to  twenty-four  hours  after  subcutaneous  inoculation. 
At  autopsy  an  extensive  oedema  of  the  subcutaneous 
tissues  about  the  seat  of  the  inoculation  is  seen,  and 
there  is  usually  a  necrotic  condition  of  the  tissues  in 
the  vicinity  of  the  point  of  puncture.  As  the  blood 
and  internal  organs  contain  the  vibrios  in  large  num- 
bers, the  infection  in  these  animals  takes,  therefore,  the 
form  of  acute,  general  septicaemia. 

Gastro-enteritis  may  be  produced  in  both  chickens 
and  guinea-pigs  by  feeding  them  with  food  in  which 
cultures  of  this  organism  have  been  mixed. 

NOTE. — More  recently,  particularly  since  the  late 
epidemic  in  Hamburg,  quite  a  number  of  curved  or 
spiral  organisms,  somewhat  like  the  cholera  spirillum, 
have  been  discovered.  For  the  descriptions  of  these 
the  reader  is  referred  to  the  current  bacteriological 
literature. 


CHAPTER  XXIV. 

Study  of  the  bacillus  anthracis,  and  the  effects  produced  by  its  inoculation 
into  animals— Peculiarities  of  the  organism  under  varying  conditions  of  sur- 
roundings. 

THE  discovery  that  the  blood  of  animals  suffering 
from  splenic  fever,  or  anthrax,  always  contained  minute 
rod-shaped  bodies  (Pollender,  1855 ;  Davaine,  1863), 
led  to  a  closer  study  of  this  disease,  and  has  resulted 
probably,  in  contributing  more  to  our  knowledge  of 
bacteriology  in  general  than  work  upon  any  of  the 
other  infectious  maladies. 

The  outcome  of  these  investigations  is  that  a  rod- 
shaped  micro-organism,  now  known  as  the  bacillus 
anthraeisy  is  always  present  in  the  blood  of  animals 
suffering  from  this  disease ;  that  this  organism  can  be 
obtained  from  the  tissues  of  these  animals  in  pure 
cultures,  and  that  these  artificial  cultures  of  the  bacillus 
anthracis  when  introduced  into  the  body  of  susceptible 
animals  can  again  produce  a  condition  identical  to  that 
found  in  the  animal  from  which  they  were  obtained. 

The  disease  is  a  true  septicaemia,  and  after  death  the 
capillaries  throughout  the  body  will  always  be  found 
to  contain  the  typical  rod-shaped  organism  in  larger  or 
smaller  numbers. 

This  organism,  when  isolated  in  pure  culture,  is  seen 
to  be  a  bacillus  which  varies  considerably  in  its  length, 
ranging  from  short  rods  of  2  to  3  /*  in  length  to  longer 
threads  of  20  to  25  p.  in  length.  In  breadth  it  is  from 


BACILLUS  ANTHEACIS.  377 

1  to  1.25  p..     Frequently  very  long  threads  made  up  of 
several  rods,  joined  end  to  end,  are  seen. 

When  obtained  directly  from  the  body  of  an  animal,  it 
is  usually  in  the  form  of  short  rods  square  at  the  ends. 
If  highly  magnified  the  ends  are  seen  to  be  a  trifle 
thicker  than  the  body  of  the  cell  and  somewhat  in- 
dented or  concave,  peculiarities  that  help  to  distinguish 
it  from  certain  other  organisms  that  are  somewhat  like 
it  morphologically.  (See  Fig.  80.) 

FIG.  80. 


Bacillus  anthracis  highly  magnified  to  show  swellings  and  concavities 
at  extremities  of  the  single  cells. 

When  cultivated  artificially  at  the  temperature  of  the 
body,  the  bacillus  of  anthrax  presents  a  series  of  very 
interesting  stages. 

The  short  rods  develop  into  long  threads,  which  may 
be  seen  twisted  or  plaited  together  after  the  manner  of 
ropes,  each  thread  being  marked  by  the  points  of  junc- 
ture of  the  short  rods  composing  it.  (Fig.  81  a,  and  6.) 

In  this  condition  it  remains  until  alterations  in  its 
surroundings,  the  most  conspicuous  being  diminution  in 
its  nutritive  supply,  favor  the  production  of  spores. 
When  this  stage  begins,  changes  in  the  protoplasm  of 
the  bacilli  may  be  noticed ;  they  become  marked  by 
irregular,  granular  bodies,  which  eventually  coalesce 
into  glistening,  oval  spores,  one  of  which  lies  in  nearly 


378  BACTERIOLOGY. 

every  segment  of  the  long  thread,  and  gives  to  the  thread 
the  appearance  of  a  string  of  glistening  beads.  (Fig. 
82.)  In  this  stage  they  remain  but  a  short  time.  The 


FIG.  81. 


!      S  $ 


Bacillus  anthracis.    Plaited  and  twisted  threads  seen  in  fresh  growing 
cultures.    X  about  400  diameters. 

chains  of  spores,  which  are  held  together  by  the  remains 
of  the  cells  in  which  they  formed,  become  broken  up, 
and  eventually  nothing  but  free  oval  spores,  and  here 
and  there  the  remains  of  mature  bacilli  which  have 
undergone  degenerative  changes  can  be  found.  In  this 

FIG.  82. 


Threads  of  bacillus  anthracis  containing  spores.    X  about  1200  diameters. 

condition  the  spores,  capable  of  resisting  deleterious 
influences,  remain  and,  unless  their  surroundings  are 
altered,  have  been  seen  to  continue  in  this  living,  though 


BACILLUS  ANTHEACIS.  379 

inactive,  condition  for  a  very  long  time.  If  again  placed 
under  favorable  conditions  each  spore  will  germinate 
into  a  mature  cell,  and  the  same  series  of  changes  will 
be  repeated  until  the  favorable  surroundings  become 
again  gradually  unfavorable  to  development,  when 
spore-formation  is  again  seen.  Spore-formation  takes 
place  only  at  temperatures  ranging  from  18°  to  43°  C., 
37.5°  C.  being  the  most  favorable  temperature.  Under 
12°  C.  they  are  not  formed.  With  this  organism  spore- 
formation  does  not  occur  in  the  tissues  of  the  living 
animal,  its  usual  condition  at  this  time  being  that  of 
short  rods.  Occasionally,  however,  somewhat  longer 
forms  may  be  seen. 

The  bacillus  of  anthrax  is  not  motile. 

GROWTH  ON  AGAR-AGAR. — The  cojonies  of  this  or- 
ganism, as  seen  upon  agar-agar,  present  a  very  typical 
appearance,  from  which  they  have  been  likened  unto 
the  head  of  Medusa.  From  a  central  point  which  is 

FIG.  83. 


Colony  of  bacittus  anthracis  on  agar-agar. 

more  or  less  dense,  consisting  of  a  felt-like  mass  of  long 
threads  matted  irregularly  together,  the  growth  continues 
outward  upon  the  surface  of  the  agar-agar.  (Fig.  83.) 
It  is  made  up  of  wavy  bundles  in  which  the  threads  are 


380  BACTERIOLOGY. 

seen  to  lie  parallel  side  by  side  or  are  twisted  in  strands 
like  those  of  a  rope — sometimes  they  have  a  plaited 
arrangement.  (See  Fig.  81.)  These  bundles  twist  about 
and  cross  in  all  directions,  and  eventually  disappear  at 
the  periphery  of  the  colony,  At  the  extreme  periphery 
of  the  colonies  it  is  sometimes  possible  to  trace  single 
bundles  of  these  threads  for  long  distances  across  the 
surface  of  the  agar-agar.  The  colony  itself  is  not  cir- 
cumscribed in  its  appearance,  but  is  more  or  less  irregu- 
larly fringed  or  ragged,  or  scalloped.  To  the  naked 
eye  they  look  very  much  like  minute  pellicles  of  raw 
cotton  that  have  been  pressed  into  the  surface  of  the 
agar-agar. 

As  the  colonies  continue  to  grow,  they  become  more 
and  more  dense,  opaque,  and  granular  and  rough  on 
the  surface.  When  touched  with  a  sterilized  needle, 
one  experiences  a  sensation  that  suggests,  somewhat, 
the  matted  structure  of  these  colonies.  The  bit  that 
may  thus  be  taken  from  a  colony  is  always  more  or  less 
ragged. 

GELATIN. — The  colonies  on  gelatin  at  the  earliest 
stages  also  present  the  same  wavy  appearance ;  but  this 
characteristic  soon  becomes  in  part  destroyed  by  the 
liquefaction  of  the  gelatin  which  is  produced  by  the 
growing  organisms.  This  allows  them  to  sink  to  the 
bottom  of  the  fluid,  where  they  lie  as  an  irregular  mass. 
Through  the  fluid  portion  of  the  gelatin  may  be  seen 
small  clumps  of  growing  bacilli,  which  look  very  much 
like  bits  of  cotton-wool. 

BOUILLON. — In  bouillon  the  growth  is  characterized 
by  the  formation  of  flaky  masses,  which  also  have  very 
much  the  appearance  of  bits  of  raw  cotton.  Microscopic 


BACILL  US  ANTHEACIS.  381 

examination  of  one  of  these  flakes  reveals  the  twisted 
and  plaited  arrangement  of  the  long  threads. 

POTATO. — It  develops  rapidly  as  a  dull,  dry,  gran- 
ular, whitish  mass,  which  is  more  or  less  limited  to  the 
point  of  inoculation.  On  potato,  at  the  temperature 
of  the  incubator,  its  spore-formation  may  easily  be  ob- 
served. 

STAB  AND  SLANT  CULTUKES. — Stab  and  slant  cul- 
tures on  agar-agar  present  in  general  the  appearances 
given  for  the  colonies,  except  that  the  growth  is  much 
more  extensive.  The  growth  is  always  more  pro- 
nounced on  the  surface  than  down  the  track  of  the 
needle. 

On  gelatin  it  causes  liquefaction,  which  begins  on  the 
surface  at  the  point  inoculated,  and  spreads  outward 
and  downward. 

It  grows  best  with  access  to  oxygen,  and  very  poorly 
when  the  supply  of  oxygen  is  interfered  with. 

Under  favorable  conditions  of  aeration,  nutrition, 
and  temperature  its  growth  is  rapid. 

Under  12°  C.  and  above  45°  C.  no  growth  occurs. 
The  temperature  of  the  body  is  most  favorable  to  its 
development. 

The  spores  of  the  anthrax  bacillus  are  very  resistant 
to  heat,  though  the  degree  of  resistance  is  seen  to  vary 
with  spores  of  different  origin.  Esmarch  found  that 
anthrax  spores  from  some  sources  would  readily  be  killed' 
by  an  exposure  of  one  minute  to  the  temperature  of 
steam,  whereas  those  from  other  sources  resisted  this 
temperature  for  longer  times,  reaching  in  some  cases  as 
long  as  twelve  minutes. 

STAINING. — The  anthrax  bacilli  stain  readily  with 
the  ordinary  aniline  dyes.  In  tissues  their  presence 

17* 


382  BACTERIOLOGY. 

may  also  be  demonstrated  by  the  ordinary  aniline  stain- 
ing fluids,  or  by  Gram's  method.  They  may  also  be 
stained  in  tissues  with  a  strong  watery  solution  of  dahlia, 
after  which  the  tissue  is  decolorized  in  2  per  cent,  sodium 
carbonate  solution,  washed  in  water,  dehydrated  in  alco- 
hol, cleared  up  in  xylol,  and  mounted  in  balsam.  This 
leaves  the  bacilli  stained,  while  the  tissues  are  decolorized ; 
or  the  tissues  may  be  stained  a  contrast  color — eosin,  for 
example — after  the  dehydration  in  alcohol,  and  before  the 
clearing  up  in  xylol.  In  this  case  they  must  be  washed 
out  again  in  alcohol  before  using  the  xylol.  In  the 
preparation  treated  in  this  way,  the  rod-shaped  organ- 
isms will  be  of  a  purple  color,  and  will  be  seen  in  the 
capillaries  of  the  tissues,  while  the  tissues  themselves 
will  be  of  a  pale  rose  color. 

INOCULATION  INTO  ANIMALS. — Introduce  into  the 
subcutaneous  tissues  of  the  abdominal  wall  of  a  guinea- 
pig  or  rabbit,  a  portion  of  a  pure  culture  of  the  bacillus 
anthracis.  In  about  forty-eight  hours  the  animal  will 
be  found  dead.  Immediately  at  the  point  of  inocula- 
tion little  or  no  reaction  will  be  noticed,  but  beyond 
this,  extending  for  a  long  distance  over  the  abdomen 
and  thorax,  the  tissues  will  be  markedly  oedematous. 
Here  and  there,  scattered  through  this  oedematous 
tissue,  small  ecchymoses  will  be  seen.  The  underlying 
muscles  are  pale  in  color.  Inspection  of  the  internal 
viscera  reveals  no  very  marked  macroscopic  changes 
except  in  the  spleen.  This  is  enlarged,  dark  in  color, 
and  soft.  The  liver  may  present  the  appearance  of 
cloudy  swelling;  the  lungs  may  be  red  or  pale-red  in 
color ;  the  heart  is  usually  filled  with  blood.  There 
are  no  other  changes  to  be  seen  by  the  naked  eye. 

Prepare  cover-slip  preparations  from  the  blood  and 


BACILLUS  ANTHRACIS.  383 

other  viscera.  They  will  all  be  found  to  contain  short 
rods  in  large  numbers.  Nowhere  can  spore- formation 
be  detected.  Upon  microscopic  examination  of  sections 
of  the  organs  which  have  been  hardened  in  alcohol,  the 
capillaries  are  seen  to  be  filled  with  the  bacilli ;  in  some 
places  closely  packed  together  in  large  numbers,  at  other 
points  fewer  in  number.  Usually  they  are  present  in 
largest  numbers  in  those  tissues  having  the  greatest 
capillary  distribution  and  at  those  points  at  which  the 
circulation  is  slowest.  They  are  moderately  evenly  dis- 
tributed through  the  spleen.  The  glomeruli  of  the 
kidneys  and  the  capillaries  of  the  lungs  are  frequently 
quite  packed  with  them.  The  capillaries  of  the  liver 
contain  them  in  large  numbers.  (Fig.  84.)  Hemor- 

FIG.  84. 


Anthrax  bacilli  in  liver  of  mouse.  X  about  450  diameters.    Bacilli  stained 
by  Gram's  method ;  tissue  stained  with  Bismarck-brown. 

rhages,  probably  due  to  rupture  of  capillaries  by  the 
mechanical  pressure  of  the  bacilli  which  are  developing 
within  them,  not  uncommonly  occur.  When  this  occurs 
in  the  mucous  membranes  of  the  alimentary  tract,  the 
blood  may  escape  through  the  mouth  or  anus  ;  when  in 
the  kidneys,  through  the  uriniferous  tubules. 


384  BACTERIOLOGY. 

Cultures  from  the  different  organs  or  from  the  oederna- 
tous  fluid  about  the  point  of  inoculation  result  in  growth 
of  the  bacillu£  anthracis. 

The  amphibia,  dogs,  and  the  majority  of  birds  are  not 
susceptible  to  this  disease.  Rats  are  difficult  to  infect. 
Rabbits,  guinea-pigs,  white  mice,  gray  house- mice,  sheep, 
and  cattle  are  susceptible.  Infection  may  occur  either 
through  the  circulation,  through  the  air-passages,  through 
the  alimentary  tract,  or,  as  we  have  just  seen,  through 
the  subcutaneous  tissues. 

EXPERIMENTS. 

Prepare  three  cultures  of  anthrax  bacilli — one  upon 
gelatin,  one  upon  agar-agar,  and  one  upon  potato. 
Allow  the  gelatin  culture  to  remain  at  the  ordinary  tem- 
perature of  the  room,  place  the  agar-agar  culture  in  the 
incubator,  and  the  potato  culture  at  a  temperature  not 
above  18°  to  20°  C.  Prepare  cover-slips  from  each 
from  day  to  day.  What  differences  are  observed? 

Prepare  two  potato  cultures  of  the  anthrax  bacillus. 
Place  one  in  the  incubator  and  retain  the  other  at  a  tem- 
perature of  from  18°  to  20°  C.  Examine  them  each 
day.  Do  they  develop  in  the  same  way  ? 

From  a  fresh  culture  of  anthrax  bacilli,  in  which 
spore-formation  is  not  yet  begun  (what  is  the  surest 
source  fro'm  which  to  obtain  non-sporebearing  anthrax 
bacilli),  prepare  a  hanging-drop  preparation ;  also  a 
cover-slip  preparation  in  the  usual  way  and  stain  it 
with  a  strong  gentian-violet  solution,  and  another 
cover-slip  preparation  which  is  to  be  drawn  through 


BACILLUS  ANTHRAC1S.  385 

the  flame  twelve  to  fifteen  times,  stained  with  aniline 
gentian-violet,  washed  off  in  iodine  solution  and  then 
in  water.  Examine  these  microscopically.  Do  they  all 
present  the  same  appearance  ?  To  what  are  the  differ- 
ences due  ? 

Do  the  anthrax  threads,  as  seen  in  a  fresh,  growing, 
hanging  drop,  present  the  same  morphological  appear- 
ance as  when  dried  and  stained  upon  a  cover-slip  ?  How 
do  they  differ  ? 

Liquefy  a  tube  of  agar-agar,  and  when  it  is  at  the 
temperature  of  40°  to  43°  C.,  add  a  very  minute  quan- 
tity of  an  anthrax  culture  which  is  far  advanced  in  the 
spore  stage.  Mix  it  thoroughly  with  the  liquid  agar- 
agar  and  from  this  prepare  several  hanging  drops  under 
strict  antiseptic  precautions,  using  the  fluid  agar-agar 
for  the  drops  instead  of  bouillon  or  salt  solution.  Select 
from  among  these  preparations  that  one  in  which  the 
smallest  number  of  spores  are  present.  Under  the 
microscope  observe  the  development  of  a  spore  into 
a  mature  cell.  Describe  carefully  the  developmental 
stages. 

Prepare  a  1  : 1000  solution  of  carbolic  acid  in  bouillon. 
Inoculate  this  with  virulent  anthrax  spores.  If  no  de- 
velopment occurs  after  two  or  three  days  at  the  tempera- 
ture of  the  thermostat,  prepare  a  solution  of  1 : 1200, 
and  continue  until  the  point  is  reached  at  which  the 
amount  of  carbolic  acid  present  just  permits  of  the  de- 
velopment of  the  spores.  When  the  proper  dilution  is 
reached  prepare  a  dozen  of  such  tubes  and  inoculate  one 
of  them  with  virulent  anthrax  spores.  As  soon  as  de- 
velopment is  well  advanced,  transfer  a  loopful  from  this 


386  BACTERIOLOGY. 

tube  into  a  second  of  the  carbolic  acid  tubes ;  when  this 
has  developed,  then  from  this  into  a  third,  etc.  After 
five  or  six  generations  which  have  been  treated  in  this 
way,  study  the  spore-production  of  the  organisms  in 
that  tube.  If  it  is  normal,  continue  to  inoculate  from 
one  carbolic  acid  tube  into  another,  and  see  if  it  is  possi- 
ble by  this  means  to  influence  in  any  way  the  production 
of  spores  by  the  organism  with  which  you  are  working. 
What  is  the  effect,  if  any? 

Prepare  two  bouillon  cultures,  each  from  one  drop  of 
blood  of  an  animal  dead  of  anthrax.  (Why  from  the  blood 
of  an  animal  and  not  from  a  culture  ?)  Allow  one  of  them 
to  grow  for  from  fourteen  to  eighteen  hours  in  the  incu- 
bator ;  allow  the  other  to  grow  at  the  same  temperature 
for  three  or  four  days.  Remove  the  first  after  the  time 
mentioned  and  subject  it  to  a  temperature  of  80°  C.  for 
thirty  minutes.  At  the  end  of  this  time  prepare  four 
plates  from  it.  Make  each  plate  with  one  drop  from 
the  heated  bouillon  culture.  At  the  end  of  three  or  four 
days  treat  the  second  tube  in  identically  the  same  way. 
How  do  the  number  of  colonies  which  developed  from 
the  two  different  cultures  compare?  Was  there  any 
difference  in  the  time  required  for  their  development 
on  the  plates? 

From  a  potato  culture  of  anthrax  bacilli  which  has 
been  in  the  incubator  for  three  or  four  days,  scrape 
away  the  growth  and  carefully  break  it  up  in  10  c.c.  of 
sterilized  physiological  salt  solution.  The  more  carefully 
it  is  broken  up  the  more  accurate  will  be  the  experiment. 
Place  this  in  a  bath  of  boiling  water  and  at  the  end  of 
one,  three,  five,  seven,  and  ten  minutes  make  a  plate 


BACILLUS  ANTHRACIS.  387 

upon  agar-agar  with  one  loopful  of  the  contents  of  this 
tube.     Are  the  results  on  the  plates  alike  ? 

Determine  the  exact  time  necessary  to  sterilize  ob- 
jects, such  as  silk  or  cotton  threads,  on  which  anthrax 
spores  have  been  dried,  by  the  steam  method  and  by  the 
hot-air  method. 

Prepare  from  the  blood  of  an  animal  just  dead  of  an- 
thrax a  bouillon  culture.  After  this  has  been  in  the 
incubator  for  from  three  to  four  hours,  subject  it  to  a 
temperature  of  55°  C.  for  ten  minutes.  At  the  end  of 
this  time  make  plates  from  it,  and  also  inoculate  a  rabbit 
subcutaneously  with  it.  What  are  the  results  ?  Are  the 
colonies  on  the  plates  in  every  way  characteristic  ? 

Inoculate  six  Erlenmeyer  flasks  of  sterile  bouillon, 
each  containing  about  35  c.c.  of  the  medium,  from  either 
the  blood  of  an  animal  just  dead  of  anthrax  or  from  a 
fresh  virulent  culture  in  which  no  spores  are  formed. 

Place  these  flasks  in  the  incubator  at  a  temperature  of 
42.5°  C.  At  the  end  of  five,  ten,  fifteen,  twenty,  twenty- 
five,  etc.,  days  remove  a  flask.  Label  each  flask  as  it  is 
taken  from  the  incubator  with  the  exact  number  of  days 
for  which  it  had  been  at  the  temperature  of  42.5°  C. 
Study  each  flask  carefully,  both  in  its  cultural  peculiar- 
ities and  its  pathogenic  properties  when  employed  on 
animals. 

Are  these  cultures  identical  in  all  respects  with  those 
that  have  been  kept  at  37°  C.? 

If  they  differ,  in  what  respect  is  the  difference  most 
conspicuous  ? 


388  BACTERIOLOGY. 

Should  any  of  the  animals  survive  the  inoculations 
made  from  the  different  cultures  in  the  foregoing  exper- 
iment, note  carefully  which  one  it  is,  and  after  ten  to 
twelve  days  repeat  the  inoculation,  using  the  same  cul- 
ture ;  if  it  again  survives,  inoculate  it  with  the  culture 
preceding  the  one  just  used  in  the  order  of  removal  from 
the  incubator  ;  if  it  still  survives,  inoculate  it  with  vir- 
ulent anthrax.  What  is  the  result  ?  How  is  the  result 
to  be  explained  ?  Do  the  cultures  which  were  made 
from  these  flasks  at  the  time  of  their  removal  from  the 
incubators  act  in  the  same  way  toward  animals  as  the 
organisms  growing  in  the  flasks  ?  Is  the  action  of  each 
of  these  cultures  the  same  for  mice,  guinea-pigs,  and 
rabbits? 

Prepare  a  2  per  cent,  solution  of  sulphuric  acid  in 
distilled  water ;  suspend  in  this  a  number  of  anthrax 
spores ;  at  the  end  of  three,  six,  and  nine  days  at  35°  C. 
inoculate  both  a  guinea-pig  and  a  rabbit.  Prepare  cul- 
tures from  this  suspension  on  the  third,  sixth,  and  ninth 
days ;  when  the  cultures  have  developed,  inoculate  a  rab- 
bit and  a  guinea-pig  from  the  culture  made  on  the  ninth 
day.  Should  the  animals  survive,  inoculate  them  again 
after  three  or  four  days  with  a  culture  made  on  the  sixth 
day.  Do  the  results  appear  in  any  way  peculiar? 


CHAPTER  XXV. 

The  most  important  of  the  organisms  found  in  the  soil— The  nitrifying  bac- 
teria—The bacillus  of  tetanus— The  bacillus  of  malignant  oedema— The  bacil- 
lus of  symptomatic  anthrax. 

BY  the  employment  of  bacteriological  methods  in  the 
study  of  the  soil  much  light  has  been  shed  upon  the  cause 
and  nature  of  the  interesting  and  momentous  biological 
phenomena  that  are  there  constantly  in  progress.  Of 
these,  the  one  that  is  of  the  greatest  importance  com- 
prises those  changes  that  accompany  the  widespread 
process  of  disintegration  and  decomposition,  to  which 
reference  has  already  been  made  (see  Chap.  I.).  This 
resolution  of  dead,  complex,  organic  compounds  into  sim- 
pler structures  that  are  assimilable  as  food  for  growing 
vegetation,  is  dependent  upon  the  activities  of  bacteria 
located  in  the  superficial  layers  of  the  ground.  It  is  not 
throughout  a  simple  process,  brought  about  by  a  single, 
specific  species  of  bacteria,  but  represents  a  series  of 
metabolic  alterations,  each  definite  step  of  which  is  most 
probably  the  result  of  the  activities  of  different  species 
or  groups  of  species,  acting  singly  or  together  (symbioti- 
cally).  Our  knowledge  upon  the  subject  is  not  suffi- 
cient to  permit  of  our  following  in  detail  the  manifold 
alterations  undergone  by  dead  organic  material  in  the 
process  of  decomposition  that  results  in  its  conversion 
into  inorganic  compounds,  with  the  formation  of  car- 
bonic acid,  ammonia,  and  water  as  conspicuous  end-pro- 
ducts. It  suffices  to  say  that,  wherever  dead  organic 


390  BACTERIOLOGY. 

matters  are  exposed  to  the  action  of  the  great  group  of 
saprophytic  bacteria,  in  which  are  found  many  different 
species,  the  alterations  through  which  they  pass  are  ulti- 
mately characterized  by  the  appearance  of  these  three 
bodies.  When  the  process  of  decomposition  occurs  in 
the  soil,  however,  it  does  not  cease  at  this  point,  but  we 
find  still  further  alterations — alterations  concerning  more 
particularly  the  ammonia.  This  change  in  ammonia  is 
characterized  by  the  products  of  its  oxidation,  viz.,  by 
the  formation  of  nitrous  and  nitric  acids  and  their  salts ; 
it  is  not  a  result  of  the  direct  action  of  atmospheric 
oxygen  upon  the  ammonia,  but  occurs  through  the 
instrumentality  of  a  special  group  of  saprophytes  known 
as  the  nitrifying  organisms.  They  are  found  in  the 
most  superficial  layers  of  the  ground,  and  though  more 
common  in  some  places  than  in  others,  they  are,  never- 
theless, present  over  the  entire  earth's  surface.  The 
most  conspicuous  example  of  the  functional  activity  of 
this  specific  form  of  soil  organism  is  that  seen  in  the 
immense  saltpetre  beds  of  Chili  and  Peru,  where,  through 
the  activities  of  these  microscopic  plants,  nitrates  are  pro- 
duced from  the  ammonia  of  the  faecal  evacuations  of 
sea-fowls  in  such  enormous  quantities  as  to  form  the 
source  of  supply  of  this  article  for  the  commercial  world. 
A  more  familiar  example,  though  hardly  upon  such  a 
great  scale,  is  that  seen  in  the  decomposition  and  subse- 
quent nitrification  of  the  organic  matters  of  sewage  and 
other  impure  waters,  in  the  process  of  purification  by 
filtration  through  the  soil ;  a  process  in  which  it  is  pos- 
sible to  follow,  by  chemical  means,  the  organic  matters 
from  their  condition  as  such,  through  their  conspicuous 
modifications  to  their  ultimate  conversion  into  ammonia, 
nitrous  and  nitric  acids.  In  fact  the  same  breaking 


NITRIFYING  BACTERIA.  391 

down  and  building  up,  resulting  ultimately  in  nitrifica- 
tion, occurs  in  all  nitrogenous  matters  that  are  thrown 
upon  the  soil  and  allowed  to  decay.  It  is  largely  through 
this  means  that  growing  vegetation  obtains  the  nitro- 
gen necessary  for  the  nutrition  of  its  tissues,  and  when 
viewed  from  this  standpoint  we  appreciate  the  impor- 
tance of  this  process  to  all  life,  animal  as  well  as  vege- 
table, upon  the  earth. 

These  very  important  and  interesting  nitrifying  or- 
ganisms, of  which  there  appear  to  be  several,  have  been 
subjected  to  considerable  study  and  are  found  to  possess 
peculiarities  of  sufficient  interest  to  justify  a  more  or 
less  detailed  description.  For  a  long  time  all  efforts  to 
isolate  them  from  the  soils  in  which  they  were  believed 
to  be  present,  and  to  cultivate  them  by  the  processes 
commonly  employed  in  bacteriological  work,  resulted  in 
failure,  and  it  was  not  until  it  was  found  that  the  ordi- 
nary methods  of  bacteriological  research  were  in  no  way 
applicable  to  the  study  of  these  bacteria  that  other,  and 
ultimately  successful,  methods  were  devised.  By  these 
special  devices  nitrifying  bacteria,  capable  of  oxidizing 
ammonia  to  nitric  acid,  have  been  isolated  and  culti- 
vated, and  the  more  important  of  their  biological  pecu- 
liarities recorded  by  Winogradsky  in  Switzerland,  by 
G.  C.  and  P.  F.  Frankland  in  England,  and  by  Jordan 
and  Richards  in  this  country.  From  the  similarity  of 
the  properties,  given  by  these  several  observers,  of  the 
nitrifying  organisms  isolated  by  them,  it  seems  likely 
that  they  have  all  been  working  with  either  the  same 
organism  or  very  closely  allied  species. 

The  organism  generally  known  as  the  nitro-monas  of 
Winogradsky  is  a  short,  oval,  and  frequently  almost 
spherical  cell.  It  divides  as  usual  for  bacteria,  but 


392  BACTERIOLOGY. 

there  is  little  tendency  for  the  daughter  cells  to  adhere 
together  or  to  form  chains.  In  cultures  they  are  com- 
monly massed  together,  by  a  gelatinous  material,  in  the 
form  of  zoogloea  They  do  not  form  spores,  and  are 
probably  not  motile,  though  Winogradsky  believes  he 
has  occasionally  detected  them  in  active  motion.  As 
has  been  stated,  they  do  not  grow  upon  the  ordinary 
nutrient  media,  and  cannot,  therefore,  be  isolated  by 
the  means  commonly  employed  in  separating  different 
species  of  bacteria.  The  most  astonishing  property  of 
this  organism  is  its  ability  to  grow  and  perform  its  spe- 
cific fermentative  function  in  solution  absolutely  devoid 
of  organic  matter.  It  is  believed  to  be  able  to  obtain 
its  necessary  carbon  from  carbonic  acid.  For  its  isola- 
tion and  cultivation  Winogradsky  recommends  the  fol- 
lowing solution  : 

Ammonium  sulphate 1    gramme. 

Potassium  phosphate 1         " 

Pure  water 1000  c.c. 

To  each  flask  containing  100  c.c.  of  this  fluid  is  added 
from  0.5  to  1.0  gramme  of  basic  magnesium  carbonate 
suspended  in  a  little  distilled  water  and  sterilized  by 
boiling.  One  of  the  flasks  is  then  to  be  inoculated  with 
a  minute  portion  of  the  soil  under  investigation,  and 
after  four  to  five  days  a  small  portion  is  to  be  withdrawn 
by  means  of  a  capillary  pipette  from  over  the  surface 
of  the  layer  of  magnesium  carbonate  and  transferred  to 
a  second  flask,  and  similarly  after  four  or  five  days  from 
this  to  a  third  flask,  and  so  on.  As  this  medium  does 
not  offer  conditions  favorable  to  the  growth  of  bacteria 
requiring  organic  matter  for  their  development,  those 
that  were  originally  introduced  with  the  soil  quickly 
disappear,  and  ultimately  only  the  nitrifying  organisms 


NITRIFYING  BACTERIA.  393 

remain.  These  are  to  be  seen  as  an  almost  transparent 
film  attached  to  the  clumps  and  granules  of  magnesium 
carbonate  on  the  bottom  of  the  flask. 

For  their  cultivation  upon  a  solid  medium  he  em- 
ploys a  mineral  gelatin,  the  gelatinizing  principle  of 
which  is  silicic  acid.  A  solution  of  from  3  to  4  per 
cent,  of  silicic  acid  in  distilled  water,  and  having  a 
specific  gravity  of  1.02,  remains  fluid  and  can  be  pre- 
served in  flasks  in  this  condition  (Kuhue).  By  the 
addition  of  certain  salts  to  such  a  solution  gelatin ization 
occurs  and  will  be  more  or  less  complete,  according  to 
the  proportion  of  salts  added.  The  salts  that  have  given 
the  best  results  and  the  method  of  mixing  them  are  as 
follows : 

f  Ammonium  sulphate .        .       .       .'  .0.4  gramme. 

a -j  Magnesium  sulphate  .       .       ...       .    0.05       " 

I  Calcium  chloride        .       .       .  »       .    trace. 

f  Potassium  phosphate .       .       .       .       .       .0.1  gramme. 

b<  Sodium  carbonate 0.6  to  0.9       " 

(.  Distilled  water 100  c.c. 

The  sulphates  and  chloride  (a)  are  mixed  in  50  c.c.  of 
the  distilled  water,  and  the  phosphate  and  carbonate  (6) 
in  the  remaining  50  c.c.,  in  separate  flasks. 

Each  flask  is  then  sterilized  with  its  contents,  which 
after  cooling  are  mixed  together.  This  represents  the 
solution  of  mineral  salts  that  is  to  be  added  to  the  silicic 
acid,  little  by  little,  until  the  proper  degree  of  consist- 
ency is  obtained  (that  of  ordinary  nutrient  gelatin).  This 
part  of  the  process  is  best  conducted  in  the  culture  dish. 
If  it  is  desired  to  separate  the  colonies,  as  in  an  ordinary 
plate,  the  inoculation  and  mixing  of  the  material  intro- 
duced must  be  done  before  gelatin  ization  is  complete;  if 
the  material  is  to  be  distributed  only  over  the  surface 


394  BACTERIOLOGY. 

of  the  medium,  then  the  mixture  must  first  be  allowed 
to  solidify. 

By  the  use  of  this  silicate-gelatin  Winogradsky  has 
isolated  from  the  gelatinous  film  in  the  bottom  of  fluids 
undergoing  nitrification,  a  bacillus  which  he  believes  to 
be  associated  with  the  nitro-monas  in  the  nitrifying 
process. 

Our  knowledge  of  these  organisms  is  as  yet  too  in- 
complete to  permit  of  a  satisfactory  description  of  all 
their  morphological  and  biological  peculiarities.  What 
has  been  said  will  serve  to  indicate  the  direction  in 
which  further  studies  of  the  subject  should  be  prose- 
cuted. 

For  further  details  the  reader  is  referred  to  the 
original  contributions.1 

In  addition  to  the  bacteria  concerned  in  putrefaction 
and  nitrification  there  are  occasionally  present  in  the 
soil  micro-organisms  possessing  disease-producing  prop- 
erties. Conspicuous  among  these  may  be  mentioned  the 
bacillus  of  malignant  oedema  (vibrion  septique  of  the 
French),  the  bacillus  of  tetanus,  and  the  bacillus  of 
symptomatic  anthrax  (Rauschbrand,  German ;  charbon 
symptomatique,  French).  It  is  sometimes  due  to  the 
presence  of  one  or  the  other  of  these  organisms  that 
wounds  to  which  soil  has  had  access  (crushed  wounds 
from  the  wheels  of  cars  or  wagons,  wounds  received  in 
agricultural  work,  etc.)  are  followed  by  such  grave  dis- 
turbances of  the  constitution. 


1  Winogradsky:  Annales  de  1'Institut  Pasteur,  tomes  iv.,  1890,  and  v.,  1891. 
Jordan  and  Richards:  Rep.  State  Board  of  Health,  Mass.,  "Purification 
of  Sewage  and  Water,"  1890,  vol.  ii.  p.  864. 

Frankland,  Q.  C.  and  P.  F. :  Proc.  Royal  Soc.  London,  1890,  xlvii. 


BACILLUS  OF  TETANUS.  395 


THE    BACILLUS    OP    TETANUS. 

In  1884  Nicolaier  produced  tetanus  in  mice  and  rab- 
bits by  the  subcutaneous  inoculation  of  particles  of 
garden  earth,  and  demonstrated  that  the  pus  produced 
at  the  point  of  inoculation  was  capable  of  reproducing 
the  disease  in  other  mice  and  rabbits.  He  did  not 
succeed  in  isolating  the  organism  in  pure  culture.  In 
1884  Carle  and  Rattone,  and  in  1886  Kosenbach,  de- 
monstrated the  infectious  nature  of  tetanus  as  it  occurs 
in  man  by  producing  the  disease  in  animals  through 
the  inoculation  of  them  with  the  secretions  from  the 
wounds  of  individuals  affected  with  the  disease.  In  1889 
Kitasato  obtained  the  bacillus  of  tetanus  in  pure  culture, 
and  described  his  method  of  obtaining  it  and  its  biological 
peculiarities  as  follows  : 

Method  of  obtaining  it.  Inoculate  several  mice  sub- 
cutaneously  with  the  secretions  from  the  wound  of  a 
case  of  typical  tetanus.  This  material  usually  contains 
not  only  tetanus  bacilli,  but  other  organisms  as  well,  so 
that  at  autopsy,  if  tetanus  results,  there  may  be  more  or 
less  of  suppuration  at  the  seat  of  inoculation  in  the  mice. 
In  order  to  separate  the  tetanus  bacillus  from  the  others 
that  are  present,  the  pus  is  smeared  upon  the  surface  of 
several  slanted  blood-serum  or  agar-agar  tubes  and 
placed  at  37°  to  38°  C.  After  twenty-four  hours  all 
the  organisms  will  have  developed  and  microscopic  ex- 
amination will  usually  reveal  the  presence  of  a  few 
tetanus  bacilli,  recognizable  by  their  shape,  viz.,  that  of 
a  small  pin,  with  a  spore  representing  the  head.  After 
forty-eight  hours  at  38°  C.  the  culture  is  subjected  to  a 
temperature  of  80°  C.  in  a  water-bath  for  from  three- 


396  BACTERIOLOGY. 

quarters  to  one  hour.  At  the  end  of  this  time,  series  of 
plates  or  Esmarch  tubes  in  slightly  alkaline  gelatin  are 
made  with  very  small  amounts  of  the  culture  and  kept 
in  an  atmosphere  of  hydrogen  (see  pages  185-191). 
They  are  then  kept  at  from  18°  to  20°  C.,  and  at  the 
end  of  about  one  week  the  tetanus  bacillus  begins  to 
appear  in  the  form  of  colonies.  After  about  ten  days 
the  colonies  should  not  only  be  examined  microscopically, 
but  each  colony  that  has  developed  in  the  hydrogen 
atmosphere  should  be  obtained  in  pure  culture  and  again 
grown  under  the  same  conditions.  The  colonies  that 
grow  only  without  oxygen,  and  which  are  composed  of 
the  pin  shaped  organisms,  must  be  tested  upon  mice. 
If  they  represent  growths  of  the  tetanus  bacillus,  the 
typical  clinical  manifestations  of  the  disease  will  be 
produced  in  these  animals. 

In  obtaining  the  organism  from  the  soil  much  diffi- 
culty is  experienced.  There  are  a  number  of  spore- 
bearing  organisms  here  that  are  facultative  in  their 
relation  to  oxygen,  and  are,  therefore,  very  difficult  to 
eliminate ;  and  there  is,  morever,  one  in  particular, 
that,  like  the  tetanus  bacillus,  forms  a  polar  spore. 
This  spore  is,  however,  less  round  and  much  more  oval 
than  that  of  the  tetanus  bacillus,  and  gives  to  the  organ- 
ism containing  it  more  the  shape  of  a  javelin  (or  dos- 
tridium,  properly  speaking)  than  that  of  a  pin,  the  char- 
acteristic shape  of  the  spore-bearing  tetanus  organism.  It 
is  non-pathogenic,  and  grows  both  with  and  without  oxy- 
gen, and  should,  consequently,  not  be  mistaken  for  the 
latter  bacillus.  It  must  also  be  borne  in  mind  that  there 
are  occasionally  present  in  the  soil  still  other  bacilli  which 
form  polar  spores,  and  which,  when  in  this  stage,  are 
almost  identical  in  appearance  with  the  tetanus  bacillus, 


BACILLUS  OF  TETANUS.  397 

but  they  will  usually  be  found  to  differ  from  it  in  their 
relation  to  oxygen,  and  they  are  also  without  disease- 
producing  properties. 

Morphology.  It  is  a  slender  rod  with  rounded  ends. 
It  may  appear  as  single  rods,  or,  in  cultures,  as  long 
threads.  It  is  motile,  though  not  actively  so.  The 


FIG.  85. 


Jl* 

2*  -v» 


'     B* 

Tetanus  bacillus.    A.  Vegetative  stage,  from  gelatin  culture.    B.  Spore 
stage,  showing  pin  shapes. 

motility  is  rendered  somewhat  more  conspicuous  by 
examining  the  organism  upon  a  warm  stage. 

At  the  temperature  of  the  body  it  rapidly  forms 
spores.  These  are  round,  thicker  than  the  cell,  and 
usually  occupy  one  of  its  poles,  giving  to  the  rod  the 
appearance  of  a  small  pin.  (Fig.  85.)  When  in  the 
spore  stage  it  is  not  motile. 

It  is  stained  by  the  ordinary  aniline  staining  reagents. 
It  remains  colored  under  the  employment  of  Gram's 
method. 

Cultural  peculiarities.  It  is  an  exquisite  anaerobe 
and  cannot  be  brought  to  development  under  the  access 
of  oxygen.  It  grows  well  in  an  atmosphere  of  pure 
hydrogen,  but  does  not  grow  under  the  influence  of 
carbonic  acid. 

It  grows  in  ordinary  nutrient  gelatin  and  agar-agar 
18 


398 


BACTERIOLOGY. 


FIG.  86 


of  a  slightly  alkaline  reaction.  Gelatin  is  slowly  lique- 
fied, with  the  coincident  production  of  a  small  amount 
of  gas.  Neither  agar-agar  nor 
blood-serum  are  liquefied  by  its 
growth. 

The  addition  to  the  media  of 
from  1.5  to  2  per  cent,  of  glucose, 
0.1  per  cent,  of  indigo-sodium- 
sulphate,  or  5  per  cent,  by  volume 
of  blue  litmus  tincture  favors  its 
growth. 

It  grows  well  in  alkaline  bouil- 
lon under  an  atmosphere  of  hy- 
drogen. 

It  may  be  cultivated  through 
numerous  generations  under  arti- 
ficial conditions  without  loss  of 
virulence. 

Appearance  of  the  colonies.  The 
colonies  on  gelatin  under  an  at- 
mosphere of  hydrogen  have,  in 
their  early  stages,  somewhat  the 
appearance  of  the  colonies  of  the 
common  bacillus  subtilis,  viz.,  they 
have  a  dense,  felt-like  centre  sur- 
rounded by  a  fringe  of  delicate 
radii.  The  liquefaction  is  so  slow 
that  the  appearance  is  retained  for 
a  relatively  long  time,  but  eventu- 
ally becomes  altered.  In  very  old 
colonies  the  entire  mass  is  made 
up  of  a  number  of  distinct  threads 


Colonies  of  the  tetanus 
bacillus  four  days  old,  made 
by  distributing  the  organ- 
isms through  a  tube  nearly 
filled  with  glucose-gelatin. 
Cultivation  under  an  at- 
mosphere of  hydrogen. 
(From  FRANKEL  and 
PFEIFPEK.) 


BACILLUS  OF  TETANUS.  399 

that  give  to  it  the  appearance  of  a  common  mould. 
(See  Fig.  86.) 

In  stab  cultures.  In  stab  cultures  made  in  tubes 
about  three-quarters  filled  with  gelatin,  growth  begins 
at  about  1.5  to  3  cm.  below  the  surface,  and  gradually 
assumes  the  appearance  of  a  cloudy  linear  mass  with 
prolongations  radiating  into  the  gelatin  from  all  sides. 
Liquefaction  with  coincident  gas  production  results,  and 
may  reach  almost  to  the  surface  of  the  gelatin. 

Relation  to  temperature  and  to  chemical  agents.  It 
grows  best  under  a  temperature  of  from  36°  to  38°  C. ; 
gelatin  cultures  kept  at  from  20°  to  25°  C.  begin  to 
grow  after  three  or  four  days.  In  an  atmosphere  of 
hydrogen  at  from  18°  to  20°  C.,  growth  does  not  usu- 
ally occur  before  one  week.  No  growth  occurs  under 
14°  C.  At  the  temperature  of  the  body,  spores  are 
formed  in  cultures  in  about  thirty  hours,  whereas  in 
gelatin  cultures  at  from  20°  to  25°  C.  they  do  not 
usually  appear  before  a  week,  when  the  lower  part  of 
the  gelatin  is  quite  fluid. 

Spores  of  the  tetanus  bacillus  when  dried  upon  bits  of 
thread  over  sulphuric  acid  in  the  desiccator  and  subse- 
quently kept  exposed  to  the  air,  retain  their  vitality  and 
virulence  for  a  number  of  months.  Their  vitality  is  not 
destroyed  by  an  exposure  of  one  hour  to  80°  C. ;  on  the 
other  hand,  an  exposure  of  five  minutes  to  100°  C.  in 
the  steam  sterilizer  kills  them.  They  resist  the  action 
of  5  per  cent,  carbolic  acid  for  ten  hours,  but  succumb 
when  exposed  to  it  for  fifteen  hours.  In  the  same  solu- 
tion, plus  0.5  per  cent,  hydrochloric  acid,  they  are  no 
longer  active  after  two  hours.  They  are  killed  when 
acted  upon  for  three  hours  by  corrosive  sublimate, 


400  BACTERIOLOGY. 

1  :1000,  and  in  thirty  minutes  by  the  same  solution  plus 
0.5  per  cent,  hydrochloric  acid. 

Action  upon  animals.  After  subcutaneous  inoculation 
of  mice  with  minute  portions  of  a  pure  culture  of  this 
organism  tetanus  develops  in  twenty-four  hours  and 
ends  fatally  in  from  two  to  three  days.  Rats,  guinea- 
pigs,  and  rabbits  are  similarly  affected,  but  only  by 
larger  doses  than  are  required  for  mice :  the  fatal  dose 
for  a  rabbit  being  from  0.3  to  0.5  c.c.  of  a  well-developed 
bouillon  culture.  The  period  of  inoculation  for  rats  and 
guinea-pigs  is  twenty-four  to  thirty  hours,  and  for  rab- 
bits from  two  to  three  days.  Pigeons  are  but  slightly, 
if  at  all,  susceptible. 

The  tetanic  convulsions  always  appear  first  in  the  parts 
nearest  the  seat  of  inoculation  and  subsequently  become 
general. 

At  autopsies  upon  animals  that  have  succumbed  to 
inoculations  with  pure  cultures^  of  the  tetanus  bacillus 
there  is  little  to  be  seen  by  either  macroscopic  or  micro- 
scopic examination,  and  cultures  from  the  seat  of  inocu- 
lation are  usually  negative,  in  so  far  as  finding  the 
tetanus  bacillus  is  concerned.  At  the  seat  of  inoculation 
there  is  usually  only  a  hypersemic  condition.  In  uncom- 
plicated cases  there  is  no  suppuration.  The  internal  or- 
gans do  not  present  any  change,  and  culture  methods  of 
examination  show  them  to  be  free  from  bacteria.  The 
death  of  the  animal  results  from  the  absorption  of  a  solu- 
ble poison,  either  produced  by  the  bacteria  at  the  seat  of 
inoculation  or,  which  seems  more  probable,  produced  by 

1  Animals  and  human  beings  that  have  become  infected  with  this  organism 
in  the  natural  way  commonly  present  a  condition  of  suppuration  at  the  site 
of  infection  ;  this  is  probably  not  due,  however,  to  the  tetanus  bacillus,  but 
to  other  bacteria  that  have  also  gained  access  to  the  wound  at  the  time  of 
infection. 


BACILLUS  OF  TETANUS.  401 

the  bacteria  in  the  culture  from  which  they  are  obtained 
and  introduced  with  them  into  the  tissues  of  the  animal 
at  the  time  of  the  inoculation.  In  support  of  the  latter 
hypothesis :  Mice  have  been  inoculated  with  pure  cul- 
tures of  this  organism  ;  after  one  hour  the  point  at 
which  the  inoculation  was  made  was  excised  and  the 
tissues  cauterized  with  the  hot  iron ;  notwithstanding 
the  short  time  during  which  the  organisms  were  in  con- 
tact with  the  tissues  and  the  subsequent  radical  treat- 
ment, the  animals  died  after  the  usual  interval  and  with 
the  regular  symptoms  of  tetanus. 

The  poison  produced  by  the  tetanus  bacillus,  and  to 
which  the  symptoms  of  the  disease  are  due,  has  been 
isolated  and  subjected  to  detailed  study ;  some  of  its 
peculiarities,  as  given  by  Kitasato,  are  as  follows  :* 

"When  cultures  of  this  organism  are  robbed  of  their 
bacteria  by  filtration  through  porcelain,  the  filtrate  con- 
tains the  soluble  poison  and  is  capable,  when  injected 
into  animals,  of  causing  tetanus. 

"  Inoculations  of  other  animals  with  bits  of  the  organs 
of  the  animal  dead  from  the  action  of  the  tetanus  poison 
produce  no  result ;  but  similar  inoculations  with  the 
blood  or  with  the  serous  exudate  from  the  pleural  cavity 
always  result  in  the  appearance  of  tetanus.  The  poison 
is,  therefore,  largely  present  in  the  circulating  fluids. 

"  The  greatest  amount  of  poison  is  produced  by  culti- 
vation in  fresh  neutral  bouillon  of  a  very  slightly  alka- 
line reaction. 

"  The  activity  of  the  poison  is  destroyed  by  an  expo- 
sure of  one  and  one-half  hours  to  55°  C. ;  of  twenty 
minutes  to  60°  C. ;  and  of  five  minutes  to  65°  C. 

i  Zeitschr.  fur  Hygiene,  1891,  Bd.  x.  p.  267. 


402  BACTERIOLOGY. 

"  By  drying  at  the  temperature  of  the  body  under 
access  of  air  the  poison  is  destroyed,  but  by  drying  at 
the  ordinary  temperature  of  the  room,  or  at  this  tem- 
perature in  the  desiccator  over  sulphuric  acid,  it  is  not 
destroyed. 

"  Diffuse  daylight  diminishes  the  intensity  of  the 
poison.  Its  intensity  is  preserved  for  a  much  longer 
time  when  kept  in  the  dark. 

"Direct  sunlight  robs  it  of  its  poisonous  properties 
in  from  fifteen  to  eighteen  hours. 

"  Its  activity  is  not  diminished  by  diluting  a  fixed 
amount  with  water  or  nutrient  bouillon. 

"  Mineral  acids  and  strong  alkalies  lessen  its  inten- 
sity." 

The  chemical  nature  of  this  poison  is  not  positively 
known,  but  according  to  the  recent  observations  of  Brie- 
ger  and  Cohn  it  is  not  to  be  classed  with  the  albumins  in 
the  sense  in  which  the  word  is  commonly  used.  When 
obtained  in  a  pure,  concentrated  form  its  toxic  proper- 
ties are  seen  to  be  altered  by  acids,  by  alkalies,  by  sul- 
phuretted hydrogen,  and  by  temperatures  above  70°  C. 
Even  when  carefully  protected  from  light,  moisture,  and 
air  it  gradually  becomes  diminished  in  strength.  When 
freshly  prepared  by  the  methods  of  the  authors  just 
cited  its  potency  is  almost  incredible,  0.000,05  milli- 
gramme beinsj  sufficient  to  cause  fatal  tetanus  in  a  mouse 
weighing  fifteen  grammes. 

THE   BACILLUS   OF   MALIGNANT   (EDEMA. 

The  bacillus  of  malignant  oedema,  also  known  as  the 
vibrion  septique,  is  another  pathogenic  form  almost  every- 
where present  in  the  soil.  In  certain  respects  it  is  a 


BACILLUS  OF  MALIGNANT  (EDEMA. 


403 


little  like  the  bacillus  of  anthrax,  and  was  at  one  time 
confounded  with  it,  but  it  differs  in  the  marked  pecu- 
liarity of  being  a  strict  anaerobe.  It  was  first  observed 
by  Pasteur,  but  it  was  not  until  later  that  Koch,  Li- 
borius,  Kitt,  and  others,  described  its  peculiarities  in 
detail,  It  can  usually  be  observed  by  inserting  under 
the  skin  of  rabbits  or  guinea-pigs  small  portions  of 
garden  earth,  street  dust,  or  decomposing  organic  sub- 
stances. There  results  a  widespread  oedema,  with  more 
or  less  of  gas  production  in  the  tissues.  In  the  oedema- 
tous  fluid  about  the  seat  of  inoculation  the  organism 
under  consideration  may  be  detected.  (Fig.  87,  A.) 

FIG.  87. 


<ra 


Bacillus  of  malignant  oedema. 

A.  Bacilli  in  short  and  long  threads  in  cedematous  fluid  from  site  of  inocu- 
lation of  guinea-pig.    (After  KOCH.) 

B.  Spore  stage  of  the  organism ;  from  culture. 


It  is  a  rod  of  about  3  to  3.5  p.  long  and  from  1  to 
1.1  IJL  thick,  i.  e.,  it  is  about  as  long  as  the  bacillus 
anthracis,  but  is  a  trifle  more  slender.  It  is  usually 
found  in  pairs,  joined  end  to  end,  but  may  occur  as 
longer  threads;  particularly  is  this  the  case  in  cultures. 


404 


BACTERIOLOGY. 


When  in  pairs,  the  ends  that  approximate  are  squarely 
cut,  while  the  distal  extremities  are  rounded.  When 
occurring  singly,  both  ends  are  round- 
ed. (How  does  it  differ  in  this  respect 
from  the  bacillus  anthracisf)  It  is 
slowly  motile  and  its  flagella  are  located 
both  at  the  ends  and  along  the  sides 
of  the  rod.  It  forms  spores  that  are 
usually  located  in  or  near  the  middle 
of  the  body  of  the  cell.  These  may 
cause  a  swelling  of  the  cell  at  the  point 
at  which  they  are  located  and  give  to 
it  a  more  or  less  oval,  spindle,  or  lozenge 
shape.  (Fig.  87,  B.) 

It  is  a  strict  anaerobe,  growing  on 
all  the  ordinary  media,  but  not  under 
the  access  of  oxygen.  It  grows  well 
in  a  hydrogen  atmosphere.  It  causes 
liquefaction  of  gelatin. 

In  tubes  containing  about  20  to  30 
c.c.  of  gelatin  that  has  been  liquefied, 
inoculated  with  a  small  amount  of  the 
culture,  and  then  rapidly  solidified  in 
ice-water,  growth  appears  in  the  form 
of  isolated  colonies  at  or  near  the  bot- 
tom of  the  tube  in  from  two  to  three 
colonies  of  the  davs  at  20°  C.  These  colonies,  when 

bacillus  of  malignant         '  *-*"-„  T 

cedema  in  deep  ge la-  of  from  0.5  to  1  mm.  in  diameter,  ap- 


tin  culture.     (After  pear  as  Yittle  spheres  filled  with  clear 

-    * 


FRANKEL  and  PFEIF- 

FEE.) 


. 

liquid,  and  are  difficult,  for  this  reason, 
to  detect.     (Fig.  88.) 

As  they  gradually  increase  in  size  the   contents    of 
the    spheres    become  cloudy  and  are    marked    by  fine 


BACILLUS  OF  MALIGNANT  (EDEMA.          405 

radiating  stripes,  easily  to  be  detected  with  the  aid  of 
a  small  hand-lens.  In  deep-stab  cultures  in  agar-agar 
and  gelatin,  development  only  occurs  along  the  track  of 
puncture  at  a  distance  below  the  surface.  Growth  is 
frequently  accompanied  by  the  production  of  gas-bubbles. 

It  causes  rapid  liquefaction  of  blood-serum  with  pro- 
duction of  gas-bubbles,  and  in  two  or  three  days  the 
entire  medium  may  have  become  converted  into  a  yel- 
lowish, semi-fluid  mass. 

The  most  satisfactory  results  in  the  study  of  the 
colonies  are  obtained  by  the  use  of  plates  of  nutrient 
agar-agar,  kept  in  a  chamber  in  which  all  oxygen  has 
been  replaced,  by  hydrogen.  The  colonies  appear  as 
dull-whitish  points,  irregular  in  outline,  and  when 
viewed  with  a  low-power  lens  are  seen  to  be  marked 
by  a  network  of  branching  and  interlacing  lines  that 
radiate  in  an  irregular  way  from  the  centre  toward  the 
periphery. 

It  grows  well  at  the  ordinary  temperature  of  the 
room,  but  reaches  its  highest  development  at  the  tem- 
perature of  the  body. 

It  stains  readily  with  the  ordinary  aniline  dyes.  It 
is  decolorized  when  treated  by  Gram's  method. 

Pathogenesis.  The  animals  that  are  known  to  be 
susceptible  to  inoculation  with  this  organism  are  man, 
horses,  calves,  dogs,  goats,  sheep,  pigs,  chickens,  pigeons, 
rabbits,  guinea-pigs,  and  mice.  Cases  are  recorded  in 
which  men  and  horses  have  developed  the  disease  after 
injuries,  doubtless  due  to  the  introduction  into  the 
wound,  at  the  time,  of  soil  or  dust  containing  the 
organism. 

If  one  introduce  into  a  pocket  beneath  the  skin  of  a 
susceptible  animal  about  as  much  garden  earth  as  can 

18* 


406  BACTERIOLOGY. 

be  held  upon  the  point  of  a  penknife,  the  animal  fre- 
quently dies  in  from  twenty-four  to  forty-eight  hours. 
The  most  conspicuous  result  found  at  autopsy  is  a  wide- 
spread cedema  at  and  about  the  seat  of  inoculation.  The 
oedematous  fluid  is  at  places  clear,  while  again  it  may 
be  marked  with  blood ;  it  is  usually  rich  in  bacilli  (Fig. 
87,  A)  and  contains  gas-bubbles.  Of  the  internal  organs 
only  the  spleen  shows  much  change.  It  is  large,  dark 
in  color,  and  contains  numerous  bacilli.  If  the  autopsy 
be  made  immediately  after  death,  bacilli  are  not  com- 
monly found  in  the  blood  of  the  heart,  but  if  deferred 
for  several  hours  the  organisms  will  be  found  in  this 
locality  also,  a  fact  that  speaks  for  their  multiplication 
in  the  body  after  death.  At  the  moment  of  death  they 
are  present  in  all  the  internal  viscera  and  on  the  serous 
surfaces  of  the  organs. 

Of  all  animals  mice  are  probably  the  most  susceptible 
to  the  action  of  this  organism,  and  it  is  not  rare  to  find 
the  organisms  in  the  heart's  blood,  even  immediately  after 
death.  They  die,  as  a  result  of  these  inoculations,  in 
from  sixteen  to  twenty  hours. 

Where  pure  cultures  are  used  for  inoculation  a  rela- 
tively large  amount  must  be  employed,  and  it  should  be 
introduced  into  a  deep  pocket  in  the  subcutaneous  tis- 
sues some  distance  from  the  surface.  In  continuing  the 
inoculations  from  animal  to  animal  small  portions  of 
organs  or  a  few  drops  of  the  oedema-fluid  should  be 
used.  The  inoculation  may  also  be  successfully  made 
by  introducing  into  a  pocket  in  the  skin  bits  of  steri- 
lized thread  or  paper  upon  which  cultures  have  been 
dried. 

The  methods  for  obtaining  the  organism  in  pure  cul- 
ture, from  the  cadaver  of  an  animal  dead  from  inocula- 


BACILLUS  OF  SYMPTOMATIC  ANTHRAX.      407 

tion,  are  in  all  essential  respects  the  same  as  those  given 
for  obtaining  cultures  from  tissues  in  general,  but  it 
must  be  remembered  that  the  organism  is  a  strict  anae- 
robe, and  will  not  grow  under  the  influence  of  oxygen 
(see  methods  of  cultivating  anaerobic  species). 

In  certain  superficial  respects  this  bacillus  suggests 
bacillus  anthracis,  but  differs  from  it  in  so  many  impor- 
tant details  that  there  is  no  excuse  for  confounding  the 
two. 

NOTE. — From  what  has  been  said  of  this  organism, 
what  are  the  most  important  differential  points  between 
it  and  bacillus  anthracis  ?  Inoculate  several  mice  with 
small  portions  of  garden  earth  and  street  dust.  Iso- 
late the  organism  that  agrees  most  nearly  with  the 
description  here  given  for  the  bacillus  of  malignant 
oedema.  Compare  its  morphological,  biological,  and 
pathogenic  peculiarities  with  those  of  bacillus  anthra- 
cis under  similar  circumstances. 

Still  another  pathogenic  organism  that  may  be  present 
in  the  soil  is 


THE   BACILLUS   OF   SYMPTOMATIC   ANTHRAX ; 

bacterie  du  charbon  symptomatique  (French) ;  Bacillus 
des  Rauschbrand  (German).  It  is  the  organism  con- 
cerned in  the  production  of  the  disease  of  young  cattle 
and  sheep  commonly  known  as  "  black  leg,"  "  quarter 
evil,"  and  "  quarter  ill,"  a  disease  that  prevails  in  cer- 
tain localities  during  the  warm  months,  and  which  is 
characterized  by  a  peculiar  emphysematous  swelling  of 
the  muscular  and  subcutaneous  cellular  tissues  over  the 


408  BACTERIOLOGY. 

quarters.  The  muscles  and  cellular  tissues  at  the  points 
affected  are  seen  on  section  to  be  saturated  with  bloody 
serum,  and  the  muscles,  particularly,  are  of  a  dark, 
almost  black  color.  In  these  areas,  in  the  bloody  transu- 
dates  of  the  serous  cavities,  in  the  bile,  and,  after  death, 
in  the  internal  organs,  the  organism  to  be  described  can 
always  be  detected.  It  is  manifest  from  this  that  the 
soil  of  localities  over  which  infected  herds  are  grazing 
may  readily  become  contaminated  through  a  variety  of 
channels,  and  thus  serve  as  a  source  of  further  dissemi- 
nation of  the  disease. 

The  organism  was  first  observed  by  Feser,  and  sub- 
sequently by  Bollinger  and  others.  The  most  complete 
description  of  its  morphological  and  biological  pecu- 
liarities is  that  of  Kitasato  (Zeitschr.  filr  Hygiene,  Bd. 
vi.  p.  105  ;  Bd.  viii.  p.  55).  The  following  is  from 


FIG.  89. 


Y* 


A  B 

Bacillus  of  symptomatic  anthrax.    (After  KITASATO.) 

A.  Vegetating  forms  from  a  gelatin  culture.    B.  Spore  forms  from  an  agar- 
agar  culture. 


Kitasato's  contributions  :  It  is  an  actively  motile  rod 
of  about  3  to  5  p.  long  by  0.5  to  0.6  p.  thick.  It  is 
rounded  at  its  ends,  and,  as  a  rule,  is  seen  singly,  though 
now  and  then  pairs  joined  end  to  end  may  occur.  It 
has  no  tendency  to  form  very  long  threads.  (Fig.  89  A.) 


BACILLUS  OF  SYMPTOMATIC  ANTHRAX.     409 


FIG.  90. 


It  forms  spores,  and  when  in  this  stage  is  seen  to  be 
slightly  swollen  at  or  near  one  of  its  poles,  the  location  in 
which  the  spore  usually  appears.    (Fig.  89, 
B.)     It  is  conspicuously  prone  to  undergo 
degenerative  changes,  and  involution  forms 
are  commonly  seen,  not  only  in  fresh  cul- 
tures, but  in  the  tissues  of  affected  ani- 
mals as  well. 

Though  actively  motile  when  in  the 
vegetative  stage,  it  loses  this  property  and 
becomes  motionless  when  spores  are  form- 
ing. 

It  is  strictly  anaerobic  and  cannot  be 
cultivated  in  an  atmosphere  in  which  oxy- 
gen is  present.  It  grows  best  under  hy- 
drogen, and  does  not  grow  under  carbonic 
acid. 

The  media  most  favorable  to  its  growth 
are  those  containing  glucose  (1.5  to  2  per 
cent.),  glycerin  (4  to  5  per  cent.),  or  some 
other  reducing  body,  such  as  indigo-so- 
dium-sulphate, sodium  formate,  etc. 

When  cultivated  upon  gelatin   plates 
in    an   atmosphere  of  hydrogen   the  col- 
onies appear  as  irregular,  slightly  lobu-     Colonies  of  the 
lated  masses.     After  a  short  time  lique-  bacillus  of  symp- 

n     ,.  n     .1  i    ..  1,1       tomatic    anthrax, 

faction   of   the   gelatin   occurs   and   the  in   deep   gelatin 
colony  presents  a  dark,  dense,  lobulated  culture.    (After 

,,     .  FRANKEL  and 

and    broken    centre,    surrounded    by    a  PFEIFFER.) 
much     more     delicate    fringe-like    zone. 

When  distributed  through  a  deep  layer  of  liquefied 
gelatin  that  is  subsequently  caused  to  solidify,  colonies 
develop  at  only  the  lower  portions  of  the  tube.  The 


410  BACTERIOLOGY. 

single  colonies  appear  as  discrete  globules  that  cause 
rapid  liquefaction  of  the  gelatin  and  ultimately  coalesce 
into  irregular,  lobulated,  liquid  areas.  In  some  of  the 
larger  colonies  an  ill-defined,  concentric  arrangement  of 
alternate  clear  and  cloudy  zones  can  be  made  out. 
(Fig.  90.) 

In  deep-stab  cultures  in  gelatin,  growth  begins  after 
about  two  or  three  days'  at  20°  to  25°  C.  It  begins 
usually  at  .about  one  or  two  centimetres  below  the  sur- 
face and  causes  slow  liquefaction  at  and  around  the  track 
of  its  development.  During  the  course  of  its  growth 
gas-bubbles  are  produced. 

In  deep-stab  cultures  in  agar-agar  at  37°  to  38°  C. 
growth  begins  in  from  twenty-four  to  forty-eight  hours, 
also  at  about  one  or  two  centimetres  below  the  surface, 
and  is  accompanied  by  the  production  of  gas-bubbles. 
There  is  produced  at  the  same  time  a  peculiar,  penetrat- 
ing odor  somewhat  suggestive  of  that  of  rancid  butter. 
Under  these  conditions  spores  are  formed  after  about 
thirty  hours. 

It  grows  well  in  bouillon  of  very  slightly  acid  reac- 
tion under  hydrogen,  but  does  not  retain  its  virulence 
for  so  long  a  time  as  when  cultivated  upon  solid  media. 
In  this  medium  it  develops  in  the  form  of  white  floc- 
culi  that  sink  ultimately  to  the  bottom  of  the  glass  and 
leave  the  supernatant  fluid  quite  clear.  If  the  vessel  be 
now  gently  shaken  these  delicate  flakes  are  distributed 
homogeneously  through  it.  In  bouillon  cultures  there 
is  often  seen  a  delicate  ring  of  gas-bubbles  around  the 
point  of  contact  of  the  tube  and  the  surface  of  the 
bouillon.  There  is  produced  also  a  peculiar  penetrating 
sour  or  rancid  odor. 

It  grows  best  at  the  body  temperature,  i.  e.,  from  37° 


BAGILL US  OF  SYMPTOMATIC  ANTHRAX.      41 1 

to  38°  C.,  but  can  also  be  brought  to  development  at 
from  16°  to  18°  C.  Under  14°  C.  no  growth  is  seen. 
Spore  formation  appears  much  sooner  at  the  higher  than 
at  the  lower  temperatures.  When  its  spores  are  dried 
upon  bits  of  thread  in  the  desiccator  over  sulphuric 
acid  and  then  kept  under  ordinary  conditions  they  retain 
their  vitality  and  virulence  for  many  months.  Similarly, 
bits  of  flesh  from  the  affected  areas  of  animals  dead  of 
this  disease,  when  completely  dried,  are  seen  to  retain 
the  power  of  reproducing  the  disease  for  a  long  time. 
The  spores  are  tolerably  resistant  to  the  influence  of 
heat :  when  subjected  to  a  temperature  of  80°  C.  for 
one  hour  their  virulence  is  not  affected,  but  an  exposure 
to  100°  C.  for  five  minutes  completely  destroys  them. 
They  are  also  seen  to  be  somewhat  resistant  to  the 
action  of  chemicals  :  when  exposed  to  5  per  cent,  car- 
bolic acid  they  retain  their  disease-producing  properties 
for  about  ten  hours,  whereas  the  vegetative  forms  are 
destroyed  in  from  three  to  five  minutes ;  in  corrosive 
sublimate  solution  of  the  strength  of  1  : 1000  the  spores 
are  killed  in  two  hours. 

When  gelatin  cultures  are  examined  microscopically 
the  organisms  are  usually  seen  as  single  rods  with 
rounded  ends.  When  cultivated  in  agar-agar  at  a 
higher  temperature  spores  are  formed  after  a  short 
time;  the  spores  are  oval,  slightly  flattened  on  their 
sides,  thicker  than  the  bacilli,  and,  as  stated,  frequently 
occupy  a  position  inclining  to  one  of  the  poles  of  the 
bacillus,  though  they  are  as  often  seen  in  the  middle. 
The  bacillus  containing  a  spore  has  usually  a  clubbed  or 
spindle  shape. 

It  stains  readily  with  the  ordinary  aniline  dyes.  It 
is  decolorized  by  Gram's  method.  Its  spores  may  be 


412  BACTERIOLOGY. 

stained   by  the   methods   usually  employed   in   spore- 
staining. 

Pathogenesis.  When  susceptible  animals,  especially 
guinea-pigs,  are  inoculated  in  the  deeper  subcutaneous 
cellular  tissues  with  pure  cultures  of  this  organism,  or 
with  bits  of  tissue  from  the  affected  area  of  another 
animal  dead  of  the  disease,  death  ensues  in  from  one  to 
two  days.  It  is  preceded  by  rise  of  temperature,  loss  of 
appetite,  and  general  indisposition.  The  seat  of  inocula- 
tion is  swollen  and  painful,  and  drops  of  bloody  serum 
may  sometimes  be  seen  exuding  from  it.  At  autopsy  the 
subcutaneous  cellular  tissues  and  underlying  muscles 
present  a  condition  of  emphysema  and  extreme  oedema. 
The  oedematous  fluid  is  often  blood-stained  and  the 
muscles  are  of  a  blackish  or  blackish-brown  color.  The 
lymphatic  glands  are  markedly  hypersemic.  The  in- 
ternal viscera  present  but  little  alteration  visible  to  the 
naked  eye.  In  the  blood-stained  serous  fluid  about  the 
point  of  inoculation  short  bacilli  are  present  in  large 
numbers.  These  often  present  slight  swellings  at  the 
middle  or  near  the  end.  They  are  not  seen  as  threads, 
but  lie  singly  in  the  tissues.  Occasionally  two  will  be 
seen  joined  end  to  end.  If  the  autopsy  be  made  imme- 
diately after  death  these  organisms  may  not  be  detected 
in  the  internal  organs,  but  if  not  made  until  after  a  few 
hours  they  will  be  found  there  also.  In  fresh  autopsies 
only  vegetative  forms  of  the  organism  may  be  found, 
but  later  (in  from  twenty  to  twenty-four  hours)  spore- 
bearing  rods  may  be  detected.  (How  does  this  compare 
with  bacillus  anthracis?)  By  successive  inoculations  of 
susceptible  animals  with  the  serous  fluid  from  the  seat 
of  inoculation  of  the  dead  animal,  the  disease  may  be 
reproduced. 


BAGILL US  OF  SYMPTOMATIC  ANTHRAX.      4]  3 

Cattle,  sheep,  goats,  guinea-pigs,  and  mice  are  sus- 
ceptible to  infection  with  this  organism,  and  present  the 
conditions  above  described ;  whereas  horses,  asses,  and 
white  rats  present  only  local  swelling  at  the  site  of  in- 
oculation. Swine,  dogs,  cats,  rabbits,  ducks,  chickens, 
and  pigeons  are,  as  a  rule,  naturally  immune  to  the 
disease. 

Though  closely  simulating  the  bacillus  of  malignant 
oedema  in  many  of  its  peculiarities,  this  organism  can, 
nevertheless,  be  readily  distinguished  from  it.  It  is 
smaller ;  it  does  not  develop  into  long  threads  in  the  tis- 
sues; it  is  more  actively  motile,  and  forms  spores  more 
readily  in  the  tissues  of  the  animal  than  does  the  bacillus 
of  malignant  oedema.  In  their  relation  to  animals  they 
also  differ,  viz.,  cattle,  while  conspicuously  susceptible 
to  symptomatic  anthrax,  are  practically  immune  toward 
malignant  oedema;  and  while  swine,  dogs,  rabbits, 
chickens,  and  pigeons  are  readily  infected  with  malig- 
nant oedema,  they  are  not,  as  a  rule,  susceptible  to 
symptomatic  anthrax.  Horses  are  affected  only  locally, 
and  not  seriously,  by  the  bacillus  of  symptomatic  an- 
thrax, but  they  are  conspicuously  susceptible  to  both 
artificial  inoculation  and  natural  infection  by  the 
bacillus  of  malignant  oedema. 

The  distribution  of  the  two  organisms  over  the  earth's 
surface  is  also  quite  different.  The  oedema  bacillus  is 
present  in  almost  all  soils,  while  the  bacillus  of  symp- 
tomatic anthrax  appears  to  be  confined  to  certain  locali- 
ties, especially  places  over  which  infected  herds  have 
been  pastured. 

A  single  attack  of  symptomatic  anthrax,  if  not  fatal, 
affords  subsequent  protection,  while  infection  with  the 
malignant  oedema  bacillus  appears  to  predispose  to  re- 
currence of  the  disease.  (Baumgarten.) 


CHAPTER  XXVI. 

Infection  and  immunity— The  types  of  infection ;  intimate  nature  of  in- 
fection—Septicaemia, toxaemia,  variations  in  infectious  processes— Immunity, 
natural  and  acquired— The  hypotheses  that  have  been  advanced  in  explana- 
tion of  immunity— Conclusions. 

AN  organism  capable  of  producing  disease  we  call 
pathogenic  or  infective,  and  the  process  by  which  it  pro- 
duces disease  we  know  as  infection.  Diseases,  therefore, 
that  depend  for  their  existence  upon  the  presence  of 
bacteria  in  the  tissues  are  infectious  diseases. 

What  is  the  intimate  nature  of  this  process  we  call 
infection  ?  Is  it  due  to  the  mechanical  presence  of 
living  bacteria  in  the  body  or  does  it  result  from  the 
deposition  in  the  tissues  of  substances  produced  by  these 
bacteria,  that  are  either  locally  or  generally  incompatible 
with  life  ?  Or,  is  the  group  of  pathological  alterations 
and  constitutional  symptoms  seen  in  these  diseases  the 
result  of  abstraction  from  the  tissues,  by  the  bacteria 
growing  in  them,  of  substances  essential  to  their  vitality? 
These  are  some  of  the  more  important  of  the  questions 
that  present  themselves  in  the  course  of  analysis  of  this 
interesting  phenomenon. 

Let  us  look  into  several  typical  infectious  diseases, 
note  what  we  find,  and  see  how  far  the  observations  thus 
made  will  aid  us  in  formulating  an  opinion.  We  begin 
with  a  study  of  those  diseases  in  which  there  is  a  general 
infection,  i.  6.,  in  which  there  is  a  general  distribution  of 
the  infective  agents  throughout  the  body.  This  group 


INFECTION  AND  IMMUNITY.  415 

comprises  the  "  septicaemias,"  and  of  them  the  disease  of 
animals  known  as  anthrax  represents  a  type  of  the 
condition.  If  the  cadaver  of  an  animal  dead  of  anthrax 
be  examined  by  bacteriological  methods  it  will  be  dis- 
covered that  there  is  present  in  all  the  organs  and 
tissues  an  organism,  a  bacillus,  of  definite  form  and 
biological  characteristics ;  and  if  the  organs,  and  tissues 
generally,  be  subjected  to  microscopic  examination  this 
same  organism  will  be  found  always  present  and  always 
located  within  the  capillaries.  At  many  points  it  will 
be  seen  crowded  in  the  capillaries  in  such  numbers  as 
to  almost,  if  not  quite,  burst  them,  and  very  commonly 
their  lumen  for  a  considerable  extent  is  entirely  occluded 
by  the  growing  bacilli.  In  such  a  case  as  this  we  might 
be  tempted  to  conclude  that  death  had  resulted  from 
mechanical  interference  with  the  capillary  circulation. 
Suppose,  however,  we  subject  the  cultures  obtained  from 
this  animal  to  conditions,  either  chemical  or  thermal, 
that  are  not  particularly  favorable  to  their  normal 
development,  and  from  time  to  time  inoculate  sus- 
ceptible animals  with  the  cultures  so  treated.  The' 
result  will  be  that,  as  we  continue  to  expose  our  cultures 
to  unfavorable  surroundings,  the  period  of  time  that  is 
required  for  them  to  cause  the  death  of  animals  will, 
in  some  cases,  gradually  become  extended,  until  finally, 
death  will  not  ensue  at  all  after  inoculation.  If,  as 
these  animals  die,  a  careful  record  of  the  conditions 
found  at  autopsy  be  kept  and  compared,  it  will  ulti- 
mately be  noticed  that  the  animals  that  die  a  longer  time 
after  inoculation  present  conditions  more  or  less  at 
variance  with  those  seen  in  the  original  animal  that  died 
more  quickly  after  having  been  inoculated.  These 
differences  usually  consist  in  a  diminution  of  the  num- 


416  BACTERIOLOGY. 

ber  of  bacilli  that  appear  upon  culture  plates  from  the 
blood  and  internal  organs,  and  in  a  lessening  in  the 
amount  of  mechanical  obstruction  offered  to  the  cir- 
culation through  plugging  of  the  capillaries  by  masses 
of  bacilli,  as  detected  by  microscopic  examination  of 
sections  of  the  organs  ;  indeed,  this  latter  condition  may 
often  have  almost,  if  not  quite,  disappeared.  We  see 
here  an  animal  dead  from  the  invasion  of  the  same 
organism  that  produced  death  in  the  first  animal,  but 
with  little  or  none  of  the  appearances  to  which  we 
were  inclined  to  attribute  the  death  of  that  animal.  It 
is  apparent  then,  that  this  organism,  with  which  we 
have  been  working,  can  destroy  the  vitality  of  an  animal 
in  a  way  other  than  by  mechanically  obstructing  its  blood- 
vessels ;  it  possesses  some  other  means  of  destroying 
life.  Possibly  its  growth  in  the  tissues  is  accompanied 
by  the  production  of  soluble  poisons,  which  when  pres- 
ent in  the  blood  are  not  compatible  with  life. 

Let  us  see  if  the  study  of  another  group  of  infections 
will  furnish  any  evidence  in  support  of  such  an  hypothe- 
sis. Introduce  into  the  subcutaneous  tissues  of  a  guinea- 
pig  a  small  amount  of  pure  culture  of  the  bacillus  of 
diphtheria.  In  three  or  four  days  the  animal  dies.  We 
proceed  with  our  autopsy  in  exactly  the  same  way  that 
we  did  with  the  animals  dead  of  anthrax,  and  will  be 
astonished  to  find  that  the  organs,  blood,  and  tissues 
generally  are  sterile,1  in  so  far  as  the  presence  of  the 
organism  with  which  the  animal  was  inoculated  is  con- 
cerned, and  by  both  culture  and  microscopic  methods 
it  is  possible  to  detect  them  only  at  the  site  of  inocula- 
tion, where  they  were  deposited.  It  is  very  evident 

1  In  by  far  the  greater  number  of  cases  this  is  true,  but  under  particular 
circumstances  there  are  exceptions. 


INFECTION  AND  IMMUNITY.  417 

that  we  have  here  a  condition  with  which  mechanical 
plugging  of  the  capillaries  could  have  had  nothing  to 
do,  for  there  are  no  organisms  in  the  blood  to  interfere 
with  its  circulation.  Our  hypothesis  then  with  regard  to 
the  condition  found  in  our  first  case  of  anthrax  is  again 
not  tenable.  Similarly,  if  an  animal  that  has  died  of 
tetanus  be  examined,  we  do  not  find  the  bacilli  in  the 
tissues  and  circulating  fluids  generally,  and,  indeed, 
often  fail  to  find  them  at  the  point  of  injury.  Plainly, 
these  fatal  results  following  upon  inoculations  with  the 
diphtheria  and  the  tetanus  bacillus,  with  their  accom- 
panying tissue  changes,  occur  from  the  presence  of  a 
something  that  cannot  be  detected  by  either  cultural 
or  microscopic  methods,  and  this  something  can  be 
only  a  soluble  substance  that  is  produced  by  the 
growing  bacteria  at  the  site  of  inoculation,  gains  ac- 
cess to  the  circulation,  and  through  this  channel  causes 
death,  for  it  is  hardly  to  be  imagined  that  the  insignifi- 
cant wound  made  in  the  course  of  inoculation  could 
per  se  have  had  this  effect.  In  other  words,  these  latter 
animals  have  died  from  what  is  called  toxcemia  (poison 
in  the  blood),  a  condition  conspicuously  different  from 
septicaemia,  as  seen  in  our  first  animal  dead  of  anthrax. 
There  are,  again,  other  infectious  diseases,  many  of 
which  are  known  to  present  variations  from  what  might 
be  considered  a  typical  course,  that  may  still  further 
serve  to  support  the  view  that  infection  is  a  process  in 
which  the  mechanical  effect  of  organisms  in  the  cir- 
culating fluids  is  of  little  consequence.  Conspicuous 
among  these  are  the  infections  that  follow  upon  the  in- 
troduction into  the  tissues  of  susceptible  animals  of  cul- 
tures of  the  micrococcus  lameolatus  (pneumococcus),  of 
the  bacillus  of  chicken  cholera,  and  of  the  organisms  con- 


418  BACTERIOLOGY. 

cerned  in  the  production  of  the  so-called  "hemorrhagic 
septicaemias."  When  running  their  normal  course  these 
organisms  cause  typical  septicaemias  after  having  been 
introduced  into  animals,  but  often,  from  causes  not  en- 
tirely clear,  the  animals  die  with  only  local  lesions,  or 
with  but  very  few  organisms  in  the  internal  viscera.  We 
see  here  conditions  analogous  to  those  observed  in  the 
two  experiments  with  anthrax,  viz.,  we  find  a  group  of 
diseases  that  are  properly  classed  as  septicaemias,  because 
of  the  usual  general  invasion  of  the  body  by  the  organ- 
isms concerned  in  their  production,  but  which  frequently 
assume  a  purely  local  character — in  both  instances  prov- 
ing fatal  to  the  animal  infected.  From  what  we  have  seen 
it  is  manifestly  probable  that,  whether  these  diseases  be 
designated  as  septicaemias  or  septic  troubles,  or  toxaemias 
or  toxic  troubles,  death  is  produced  in  all  instances  by 
the  poisonous  products  resulting  from  the  growth  of 
the  infecting  bacteria.  In  the  case  of  typical  anthrax, 
and  other  varieties  of  septicaemia,  the  production  of  this 
poison  is  associated  with  the  general  dissemination  of 
the  organisms  throughout  the  body,  while  in  those  infec- 
tions often  referred  to  as  toxaemias,  of  which  diphtheria 
may  be  taken  as  a  type,  the  poison  is  produced  by  the 
organisms  that  remain  localized  at  the  site  of  invasion, 
and  is  from  thence  disseminated  throughout  the  body 
by  the  circulating  fluids. 

Infection  thus  far,  then,  appears  to  be  a  chemical 
process. 

Through  special  investigations  that  have  been  made 
upon  the  products  of  growth  of  certain  pathogenic  bac- 
teria, this  opinion  has  received  further  confirmation ; 
it  has  been  found  possible  by  the  use  of  appropriate 
methods  to  isolate,  from  among  the  mass  of  material  in 


INFECTION  AND  IMMUNITY.  41 9 

which  certain  of  these  organisms  have  been  artificially 
cultivated,  substances  which,  when  separated  from  the 
bacteria  by  which  they  were  produced,  possess  the  power 
of  causing  in  animals  all  the  constitutional  symptoms 
and  pathological  tissue  changes  that  are  seen  to  occur  in 
the  course  of  infection  by  the  organisms  themselves. 
In  some  instances  these  poisons,  toxins1  as  they  are  col- 
lectively called,  appear  to  be  the  direct  result  of  metabolic 
changes  brought  about  by  bacteria  in  the  medium  or 
tissues  in  which  they  may  be  developing,  i.  e.,  they  are 
products  of  nutrition  that  pass  readily  into  solution,  as 
is  conspicuously  seen  in  the  case  of  the  bacillus  of  diph- 
theria and  of  tetanus  when  under  both  artificial  cultiva- 
tion and  in  the  animal  body.  Many  bacteria  which  do 
not  possess  the  power  of  generating  or  secreting  such 
poisons  may,  nevertheless,  have  intimately  associated 
with  their  protoplasmic  bodies  poisonous  substances  that 
can  only  be  isolated  by  particular  methods ;  thus  Buch- 
ner  has  isolated  from  several  species  of  bacteria  u  bac- 
terio-proteius  "  having  the  common  properties  of  solu- 
bility in  alkalies,  resistance  to  the  boiling  temperature, 
attraction  of  leucocytes  (positive  chemotaxis),  and  pyo- 
genic  powers. 

There  is  as  yet  little  agreement  of  opinion  as  to  the 
chemical  nature  of  toxins,  but  it  is  probable  that  the 
group  comprises  different  bodies  of  the  nature  of  globu- 
lins, nucleo-alburnins,  peptones,  albumoses,  and  enzymes 
or  ferments. 

Toxic  ptomaines  are  probably  not  conspicuously  con- 
cerned in  producing  the  characteristic  symptoms  of 


1  "  Toxins  "  is  the  term  commonly  used  to  designate  amorphous  poisons  of 
a  proteid  nature ;  while  "  ptomaines"  is  the  term  used  to  signify  nitrogenous 
poisons  that  are  crystattizable. 


420  BACTERIOLOGY. 

infection,  as  they  are  absent  from  cultures  of  certain 
highly  pathogenic  bacteria. 

In  some  instances  the  production  of  the  poisonous 
principles,  even  under  artificial  conditions  of  cultiva- 
tion, is  of  a  most  astonishing  nature,  and  poisons  result 
that,  in  the  degree  of  their  toxicity,  exceed  anything 
hitherto  known  to  us.  For  instance,  the  potencies  of 
the  poisons  that  have  been  isolated  from  cultures  of  the 
bacillus  diphtherias  and  the  bacillus  of  tetanus  have  been 
carefully  determined  by  experiments  upon  animals,  and 
it  has  been  found  that  0.4  milligramme  of  the  former  is 
capable  of  killing  eight  guinea-pigs,  each  weighing  400 
grammes,  or  two  rabbits,  each  weighing  three  kilo- 
grammes (Roux  and  Yersin1) ;  and  that  0.0001  milli- 
gramme of  the  latter  will  produce  tetanus  in  a  mouse, 
with  all  the  characteristic  manifestations  of  the  disease 
(Brieger  and  Cohn2). 

In  short,  infection  may  be  best  conceived  as  a  contest 
between  the  invading  organisms  on  the  one  side  and  the 
resisting  tissues  of  the  animal  body  on  the  other,  the 
weapons  of  offense  of  the  former  being  the  poisonous 
products  of  their  growth,  the  toxins,  and  the  means  of 
defense  possessed  by  the  latter  being  substances  which 
are,  so  to  speak,  antidotal  to  these  poisons.  To  these 
substances  possessed  by  the  animal  body  for  resisting 
infection  the  name  "alexines"  has  been  given  by  Buch- 
ner,  while  the  name  "  defensive  proteids  "  is  suggested 
by  Hankin.  If  the  tissue  elements  are  not  of  sufficient 
vigor  to  neutralize  the  bacterial  poisons,  the  bacteria  are 
victorious,  and  infection  results,  while,  if  there  be  failure 
to  establish  a  condition  of  disease,  the  tissues  are  vic- 

1  Annales  de  1'Institut  Pasteur,  tome  iii.,  1889,  p.  287. 

2  Zeitschr.  fur  Hygiene  u.  Infektionskrankheiten,  1893,  Bd.  xv.,  Heft  i. 


INFECTION  AND  IMMUNITY.  421 

torious,  and  are  said  to  be  resistant  or  to  possess  immun- 
ity to  this  particular  form  of  infection. 

It  is  a  common  observation  that  certain  human  beings 
and  animals  are  more  susceptible  to  the  different  forms 
of  infection  than  are  others,  and  that  some  are  appar- 
ently not  at  all  liable  to  particular  diseases;  in  other 
words,  they  are  naturally  immune  to  the  maladies. 

Again,  it  is  often  observed  that  an  individual  or  ani- 
mal after  having  recovered  from  certain  forms  of  infection 
has  thereby  acquired  protection  against  subsequent  at- 
tacks of  like  character ;  in  other  words,  they  are  said  to 
have  acquired  immunity  to  this  trouble. 

The  problem  involving  the  explanation  of  these  inter- 
esting observations  has  afforded  material  for  reflection 
and  hypothesis  for  a  long  time,  but  it  is  only  through 
investigations  that  have  been  conducted  during  the  past 
few  years  that  it  has  met  with  anything  approaching 
reasonable  solution. 

Conspicuous  among  the  observers  who  have  endeav- 
ored to  explain  the  modus  operandi  of  immunity  may  be 
mentioned  Chauveau,  Pasteur,  Metchnikoff,  Buchner, 
Fliigge  and  ^his  pupils  (Smirnow,  Sirotinin,  Bitter,  Nut- 
tall),  Fodor,  and  Haukin,  and  in  the  following  pages  we 
will  present  briefly  the  result  of  investigations  by  these 
various  authors. 

In  1880  Chauveau1  suggested  an  explanation  for  the 
phenomenon  of  immunity  that  has  since  been  known  as 
the  retention  hypothesis.  It  is,  in  short,  as  follows  :  That 
the  immunity  commonly  seen  to  exist  in  animals  that 
have  passed  through  an  attack  of  infection,  against  a 
subsequent  outbreak  of  the  same  malady,  and  likewise 

1  Comptes-rendus,  etc.,  July,  1880,  No.  91. 
19 


422  BACTERIOLOGY. 

the  immunity  that  has  been  produced  artificially  by  vac- 
cination, exists  by  virtue  of  some  bacterial  product  that 
has  been  retained  or  deposited  in  the  tissues  of  those 
animals,  and  that  this  product  by  its  presence  prevents 
the  development  of  the  same  organisms  if  they  should 
subsequently  gain  access  to  the  body. 

Bearing  upon  this  view  the  experiments  of  Sirotinin,1 
made  with  cultures  of  various  pathogenic  bacteria,  dem- 
onstrated that,  in  so  far  as  culture  experiments  were  con- 
cerned, the  only  substances  produced  by  growing  bacteria 
that  could  be  in  any  way  inimical  to  their  further  devel- 
opment were  substances  that  gave  rise  to  alterations  in 
the  reaction  of  the  medium  in  which  they  were  develop- 
ing, i.  e.j  acids  or  alkalies  produced  by  the  bacteria 
themselves.  So  long  as  the  organisms  were  not  actually 
dead  from  exposure  to  these  substances,  correction  of  the 
abnormal  reaction  was  followed  by  further  development 
of  the  organisms.  Sirotinin  also  states  that  materials 
containing  the  products  of  growth  of  bacteria,  so  long  as 
they  are  maintained  at  a  neutral  or  only  slightly  alka- 
line reaction,  serve  very  well  as  media  upon  which  to 
cultivate  again  the  same  organism  that  produced  them, 
providing  the  nutritive  elements  have  not  been  entirely 
exhausted.  He  remarks  that,  if  in  such  a  concentrated 
form  as  we  find  the  life  products  of  bacteria  in  the 
medium  in  which  they  are  growing,  no  inhibitory  com- 
pounds beyond  acids  and  alkalies  are  to  be  detected,  it  is 
hardly  probable  that  they  are  produced  in  the  tissues  of 
the  living  animal,  and  retained  there,  to  a  degree  suffi- 
cient to  prevent  the  growth  of  bacteria  that  may  subse- 
quently gain  entrance  to  these  tissues,  after  the  disap- 

i  Zeitsch.  fur  Hygiene,  1888,  Bd.  iv. 


INFECTION  AND  IMMUNITY.  ,  423 

pearance  of  the  organisms  concerned  in  the  primary 
invasion.  On  the  other  hand,  Salmon  and  Smith,1 
Roux  and  Chamberland,2  and  others  had  demonstrated 
that  a  sort  of  immunity  against  certain  forms  of  infec- 
tion may  be  afforded  to  susceptible  animals  by  the  injec- 
tion into  their  tissues  of  the  products  of  growth  of  par- 
ticular organisms  which,  if  themselves  introduced  into 
the  animal  body,  would  produce  fatal  results.  In  the 
light  of  subsequent  experiments,  however,  the  interpre- 
tation of  this  phenomenon  is  not  that  claimed  by  the 
supporters  of  this  hypothesis. 

As  opposed  to  the  view  of  Chauveau,  Pasteur3  and 
certain  of  his  pupils  believed  that  the  immunity  fre- 
quently afforded  to  the  tissues  by  an  attack  of  infection, 
or  following  upon  vaccination  against  infection,  was  due 
rather  to  an  abstraction  from  the  tissues,  by  the  organ- 
isms that  were  concerned  in  the  primary  attack,  of  a 
something  that  is  necessary  to  the  growth  of  the  infect- 
ing organism  shotrld  it  gain  entrance  to  the  body  at  any 
subsequent  time.  This  view  is  known  as  the  exhaustion 
hypothesis. 

As  to  the  exhaustion  hypothesis  of  Pasteur,  there  is, 
as  yet,  no  evidence  whatever  for  its  support.  The  work 
of  Bitter,4  which  was  undertaken  with  the  view  of  de- 
termining if,  in  the  process  of  acquiring  immunity,  there 
occurred  this  exhaustion  from  the  tissues  of  material 
necessary  to  the  growth  of  bacteria  that  might  gain  en- 
trance to  them  at  some  later  date,  gave  only  negative 
results.  The  flesh  of  animals  in  which  immunity  had 
been  produced  contained  all  the  elements  necessary  for 

1  Proc.  of  the  Biol.  Soc.,  Washington,  D.  C.,  1886,  vol.  iii. 

2  Annales  de  Tlnstitut  Pasteur,  1888-89,  tomes  i.,  ii. 

3  Bull,  de  1'Acad.  de  Med.,  1880.        4  Zeitschr.  fur  Hygiene,  1888,  Bd.  iv. 


424  BACTERIOLOGY. 

the  growth  and  nutrition  of  the  bacteria  against  which 
the  animals  had  been  protected,  just  as  did  the  flesh  of 
non-vaccinated  animals. 

In  1884  Metchnikoff l  published  the  first  of  a  series 
of  observations  upon  the  relation  that  is  seen  to  exist 
between  certain  of  the  mesodermal  cells  of  lower  animals 
and  insoluble  particles  that  may  be  present  in  the  tissues 
of  these  animals.  The  outcome  of  these  investigations 
was  the  establishment  of  his  well-known  doctrine  of 
phagocytosis,  the  principle  of  which  is  that  the  wander- 
ing cells  of  the  animal  organism,  the  leucocytes,  possess 
the  property  of  taking  up,  rendering  inert,  and  digesting 
micro-organisms  with  which  they  may  come  in  contact 
in  the  tissues.  Metchuikoif  believed  that  in  this  way 
immunity  against  infection  may  in  many,  if  not  all,  cases 
be  explained.  He  believed  that  susceptibility  to  or  im- 
munity against  infection  was  essentially  a  matter  be- 
tween the  invading  bacteria  on  the  one  hand  and  the 
leucocytes  of  the  tissues  on  the  other.  The  success  or 
failure  of  the  leucocytes  in  protecting  the  animal  against 
infection  depends,  according  to  this  doctrine,  entirely 
upon  the  efficiency  of  the  means  possessed  by  them  for 
destroying  bacteria.  When  these  means  are  of  sufficient 
vigor  to  bring  about  the  death  of  the  bacteria,  the  tissues 
are  victorious,  but  when  the  poisons  generated  by  the 
bacteria  are  potent  to  arrest  the  phagocytic  action  of  the 
leucocytes,  then  the  tissues  succumb  and  infection  re- 
sults. 

Has  this  doctrine  of  phagocytosis,  as  advanced  by 
Metchnikoif,  stood  the  test  of  experimental  criticism  ? 
Evidence  that  has  accrued  since  the  time  of  its  sugges- 

1  Arbeiten  aus  dem  Zoologischen  Institut  der  Universitat  Wien.,  1884,  Bd.  v 
Fortschritte  der  Med.,  1884,  Bd.  ii. 


INFECTION  AND  IMMUNITY.  425 

tion  has  rendered  questionable  the  advisibility  of  its 
unconditional  adoption. 

The  first  severe  blow  that  this  theory  received  was 
given  by  Nuttall,1  in  his  work  upon  the  anti -bacterial 
action  of  the  animal  economy.  In  these  experiments 
Nuttall  showed  positively  that  the  part  played  by  the 
leucocytes  was  not  essential  to  the  destruction  of  viru- 
lent bacteria  in  the  blood  of  animals,  but  that  the  serum 
of  the  blood,  when  quite  free  from  cellular  elements,  pos- 
sessed this  power  to  a  degree  equal  to  that  of  the  blood 
when  all  the  constituent  parts  were  present.  In  the 
blood,  as  such,  phagocytosis  could  be  seen,  but,  as  a 
rule,  the  bacteria  presented  evidence  of  having  under- 
gone degenerative  changes  before  they  had  been  taken 
up  by  the  wandering  cells. 

Contrary  to  the  notions  in  existence  at  the  time, 
Traube  and  Gscheidlen,2  as  far  back  as  1874,  demon- 
strated that  considerable  quantities  of  septic  material 
could  be  injected  into  the  circulating  blood  without  ap- 
parently any  effect  upon  the  animal.  As  a  result  of 
these  experiments,  the  question  that  naturally  presented 
itself  was :  Does  the  animal  organism  possess  the  power 
of  rendering  septic  organisms  inert,  and  if  so,  to  what 
extent?  Their  further  work  showed  that  appreciable 
numbers  of  living  bacteria  could  be  injected  into  the 
circulation  of  warm-blooded  animals  without  producing 
any  noticeable  effect.  Particularly  was  this  the  case 
with  dogs.  If  they  injected  into  the  circulation  of  a  dog 
as  much  as  1.5  c.cm.  of  decomposing  fluid,  the  blood 
drawn  from  the  animal  after  from  twenty-four  to  forty- 
eight  hours  showed  no  especial  tendency  to  decompose, 

1  Zeitschrift  fur  Hygiene,  1888,  vol.  iv. 

2  Jahresbericht  der  Schlesischen  Ges.  fur  Cultur,  1874 ;  Jahr.  lii.  p.  179. 


426  BACTERIOLOGY. 

though  it  was  kept  under  observation  for  a  long  time. 
They  believed  this  power,  of  rendering  living  organisms 
inert,  to  be  possessed  by  the  circulating  blood  to  only  a 
limited  degree,  for,  after  the  injection  of  much  larger 
amounts  of  the  putrid  fluid  into  the  blood  of  the  animal, 
death  usually  ensued  in  from  twenty-four  to  forty-eight 
hours.  The  blood  drawn  from  the  animal  just  before 
death  contained  the  living  bacteria  of  putrefaction,  and 
underwent  decomposition.  They  attributed  the  germi- 
cidal  phenomenon  to  the  action  of  the  "  ozonized  oxygen 
of  the  corpuscles  of  the  blood." 

In  1882  Rauschenbach1  demonstrated  that,  in  the 
process  of  coagulation,  fibrin  was  formed  not  as  a  spe- 
cific product  of  the  action  of  the  colorless  elements  of  the 
blood  alone,  but  also  as  a  result  of  the  combined  action 
between  all  animal  protoplasm  and  healthy  blood  plasma, 
and  that  in  the  process  there  was  always  a  disintegration 
of  the  leucocytes  that  were  present.  In  1884  Groth2 
demonstrated  further  that  such  a  disintegration  of  leu- 
cocytes occurred  in  normal  circulating  blood,  though 
here  it  was  not  accompanied  by  coagulation.  The  re- 
sults of  these  observations  suggested  the  question :  Does 
such  a  disintegration  occur  when  vegetable  protoplasm  is 
introduced  into  the  blood?  For  the  purpose  of  answer- 
ing this  question,  Grohmaun,3  a  pupil  of  Alexander 
Schmidt,  undertook  to  study  the  action  of  the  circulat- 
ing blood  upon  the  vegetable  protoplasm  of  bacteria. 

He  noticed  that  clotting  of  the  blood  of  the  horse 

1  Ueber  die  Wechselwirkung  zwischen  Protoplosma  und  Blutplasma.    Dis- 
sertation, Dorpat,  1882. 

2  Ueber  die  Schicksale  der  farblosen  Elemente  in  kreisendem  Blut.    Disser- 
tation, Dorpat,  1884. 

3  Ueber  die  Einwirkung  des  zellenfreien  Blutplasma  auf  einige  pflanzliche 
Mikro-organismen.    Dissertation,  Dorpat,  1884. 


INFECTION  AND  IMMUNITY.  427 

was  very  much  accelerated  by  the  addition  to  it  of  cer- 
tain bacteria,  and  that  at  the  same  time  the  develop- 
ment of  the  bacteria  was  checked,  and  in  the  case  of 
the  pathogenic  varieties  their  virulence  was  diminished. 
This  was  particularly  the  case  when  the  anthrax  bacillus 
was  employed. 

Grohmann  seems  to  have  appreciated  the  significance 
of  this  observation,  though  he  took  no  steps  to  study  it 
more  closely.  He  remarks  that  the  system  probably 
possesses,  in  the  plasma  of  the  blood,  a  body  having 
disinfectant  properties  (loo.  cit.,  pp.  6  and  33).  This 
work,  however,  was  not  conducted  according  to  the 
more  exact  methods  of  modern  bacteriological  researcji, 
so  that  the  complete  demonstration  of  this  phenomenon 
must  be  attributed  to  Nuttall. 

Since  the  publication  of  NuttalPs  work  his  results 
have  received  confirmation  from  all  sides.  Fodor,1 
Bnchner,2  Lubarsch,3  Nissen,4  Stern,5  Prudden6  Charrin, 
Roger,7  and  others  have  continued  in  the  same  line,  and 
have  all  made  practically  the  same  observation. 

After  the  demonstration  by  Nuttall  that  the  serum  of 
the  blood  was  directly  detrimental  to  the  vitality  of 
certain  pathogenic  bacteria,  it  became  the  work  of  a 
number  of  investigators  to  determine  to  which  element 
of  the  serum  this  property  is  due,  or  if  it  is  a  function  of 
the  serum  only  as  a  whole. 

In  the  course  of  Buchner's  experiments  it  was  demon- 


Centr.  f.  Bakteriologie  u.  Parasitenkunde,  1890,  vol.  vii.,  No.  24. 
Archiv  fiir  Hygiene,  1890,  vol.  x.  parts  1  and  2. 
Centr.  f.  Bakt.  u.  Parasitenkunde,  1889,  vol.  vi.,  No.  18. 
Zeitschr.  fiir  Hygiene,  1889,  vol.  vi.  part  3. 
Zeitschr.  fiir  kliu.  Med.,  1890,  vol.  viii.  parts  1  and  2. 
N  Y.  Med.  Record,  1890,  vol.  xxxvii.,  pp.  85,  86. 
7  Soe.  de  Biol.  de  Paris. 


428  BACTERIOLOGY. 

strated  that  the  serum  was  robbed  of  this  property  by 
an  exposure  to  a  temperature  of  55°  C.  for  half  an 
hour ;  that  its  efficacy  as  a  germicide  was  not  diminished 
by  alternate  freezing  and  thawiug;  that  by  dialysis  or 
extreme  dilution  with  distilled  water,  its  germicidal 
activity  was  diminished,  or  completely  checked  ;  but  that 
an  equal  dilution  could  be  made,  if  sodium  chloride  so- 
lution (0.6-0.7  per  cent.)  was  substituted  for  the  dis- 
tilled water,  without  the  bactericidal  action  of  the  serum 
losing  any  of  its  power.  From  this  he  coucluded  that 
the  active  element  in  this  phenomenon  is  a  living  albu- 
min, an  essential  constituent  of  which  is  sodium  chloride, 
and  which,  when  robbed  of  this  salt,  either  by  dialysis 
or  dilution,  becomes  inert  in  its  behavior  toward  bacteria. 
For  this  or  these  germicidai  constituents  of  the  blood 
serum  he  suggested  the  name  "  Alexines." 

He  found,  moreover,  that  the  activity  of  the  serum 
alone  against  bacteria  was  greater  than  when  the  cellular 
elements  of  the  blood  were  present.  This  he  explains 
by  the  assumption  that  in  the  serum  alone  the  germi- 
cidal element  predominates,  whereas  in  the  blood,  as 
such,  outside  of  the  body,  it  is  still  present,  but  is  over- 
balanced by  the  nutrition  offered  by  the  disintegrated 
cellular  elements ;  so  that  here  the  nutritive  element  is 
most  conspicuous,  and  the  destructive  activity  toward 
bacteria  is  less  effectual. 

A  closer  study  of  the  nature  of  this  germicidal  ele- 
ment in  the  body  of  animals  was  made  by  Hankin  and 
Martin.1  The  former  isolated  from  the  spleen  and 
lymphatic  glands  a  body — a  globulin — which  in  solution 
possesses  germicidal  properties. 

i  British  Medical  Journal,  1890,  May  31. 


INFECTION  AND  IMMUNITY.  429 

Similar  germicidal,  ferment-like  globulins  have  been 
isolated  from  the  blood  by  Ogata,1  and  in  their  studies 
upon  tetanus,  Tizzoni  and  Cattani2  found  a  body  that 
was  antagonistic  to  the  poison  produced  by  the  organism 
of  this  disease. 

According  to  the  observations  of  Vaughan,3  the  most 
important  germicidal  or  protective  agents  possessed  by 
the  body  are  the  nucleins. 

Hankiu  believes  the  globulins  or  "  defensive  pro- 
teids"  that  he  has  discovered  and  the  albuminoid 
bodies  studied  by  Buchner  to  be  identical.  The  most 
interesting  and,  in  the  light  of  work  that  has  appeared 
since,  the  most  important,  of  Hankin's  observations  were 
not  those  upon  the  power  of  these  globulins  to  destroy 
the  vitality  of  living  organisms,  but  rather  those  upon 
the  relation  between  them  and  the  poisonous  proteid 
products  of  the  organisms.  For  example,  if  the  poison- 
ous products  of  virulent  anthrax  bacilli  be  isolated  and 
mixed  with  the  globulin  extracted  from  normal  tissues, 
the  experiments  of  Hankin  showed  a  directly  destruc- 
tive action  on  the  part  of  the  bacterial  products.  He 
found  that  the  amount  of  poisonous  albumose  produced 
by  the  attenuated  anthrax  bacilli,  that  are  employed  as 
vaccines,  was  much  less  than  that  produced  by  the  organ- 
isms possessing  full  virulence,  and  he  suggests  that  per- 
haps the  protective  influence  of  vaccinations  that  are 
practised  by  introducing  into  the  animal  the  organisms 
that  have  been  attenuated  in  virulence,  is  due  to  a 
gradual  tolerance  acquired  by  the  cells  of  the  tissues  to 
the  action  of  the  poison  when  produced  in  these  small 


1  Centr.  f.  Bakt.  u.  Parasitenkunde,  1891,  vol  ix.,  p.  599. 

2  Ibid.,  p.  685 

»  Vaughan  :  Medical  News,  1893,  May  20. 
19* 


430  BACTERIOLOGY. 

quantities  ;  in  the  same  way  that  a  tolerance  was  ac- 
quired by  the  tissues  for  the  venom  of  the  rattlesnake  in 
the  experiments  of  Sewall,1  and  similar  to  that  following 
the  injection  into  the  tissues  of  small  quantities  of 
hemialbumose,  which  in  large  amounts  rapidly  proves 
fatal. 

Of  utmost  importance  to  these  studies  of  the  blood 
and  fluids  of  the  body  are  the  experiments  of  Behring 
and  Kitasato2  upon  the  production  of  immunity  to 
tetanus.  In  their  studies  upon  the  blood  of  animals 
subjected  to  -these  experiments  it  was  found  that  it  was 
not  only  possible  to  render  animals  immune  from  this 
disease,  but  that  the  serum  of  the  blood  of  these  irn- 
munified  animals  afforded  immunity  when  injected  into 
the  peritoneal  cavity  of  other  animals  that  had  not  been 
so  protected ;  and  moreover,  that  this  serum  possesses 
curative  powers  over  the  disease  after  it  has,  in  some 
cases,  been  in  progress  for  a  time.  They  found,  further, 
that  the  serum  of  animals  that  had  been  rendered  im- 
mune to  tetanus,  when  brought  in  contact  with  the  poison 
of  tetanus,  completely  destroyed  its  poisonous  properties, 
and  that  the  serum  from  animals  or  from  human  beings 
that  do  not  possess  immunity  to  this  disease 'has  no  such 
power. 

The  demonstration  by  Behring  and  Kitasato  of  the 
fact  that  the  serum  of  an  immunified  animal  can  not 
only  confer  immunity  to  another  susceptible  animal,  but, 
in  the  case  of  tetanus,  cure  the  disease  after  it  is  already 
in  progress,  is  one  of  the  most  important  steps  that  has 
been  made  in  the  entire  field  of  study.  The  subsequent 
application  of  the  principle  involved  in  that  observation, 

1  Journal  of  Physiology,  1887,  vol.  viii.  p.  203. 

2  Behring  and  Kitasato :  Deutsche  med.  Woch.,  1890,  Bd.  xvi.  p.  1113. 


INFECTION  AND  IMMUNITY.  431 

by  Behring  and  his  colleagues,  in  their  successful  efforts  to 
devise  a  cure  for  diphtheria  in  man  has  resulted  in  one  of 
the  greatest  triumphs  of  the  day.  It  marks  an  epoch  in 
modern  scientific  medicine.  The  same  principle  has  been 
employed  for  obtaining  curative  agents  against  other 
forms  of  infection,  but  as  yet,  unfortunately,  with  in- 
different success. 

Another  hypothesis  in  explanation  of  the  immunity 
acquired  by  the  tissues  of  the  animal  organism  is  that 
advanced  by  Buchner,1  who  suggests  that  in  the  primary 
infection,  from  which  the  animal  has  recovered,  there 
has  been  produced  a  reactive  change  in  the  integral  cells 
of  the  body  that  enables  them  to  protect  themselves 
against  subsequent  inroads  of  the  same  organism. 
Though  somewhat  more  vague  at  first  glance  than  the 
other  theories  in  regard  to  this  phenomenon,  it  is,  never- 
theless, in  the  light  of  subsequent  research,  most  prob- 
ably the  correct  explanation  of  the  establishment  of 
immunity  in  many,  if  not  all,  cases.  Experiments  that 
bear  directly  upon  this  idea  have  demonstrated  that,  if 
animals  be  subjected  to  injections  of  the  poisonous 
products  of  growth  of  certain  virulent  bacteria,  they 
respond  to  this  treatment  by  more  or  less  pronounced 
constitutional  reactions,  and  that  during  this  period,  and 
for  a  short  time  following,  they  possess  protection 
against  the  invasion  of  the  virulent  bacteria  themselves. 
This  observation  has,  moreover,  not  been  confined  to 
those  cases  in  which  injections  of  the  products  of  growth 
have  been  followed  by  inoculations  with  the  bacteria  by 
which  they  were  produced,  but  what  is  still  more  in- 
teresting, and  confirmatory  of  Buchner's  view,  it  is 

1  Buchner :  Eine  neue  Theorie  iiber  Erzieiung  von  Immunitat  gegen  In- 
fektionskrankheiten.    Munich,  1883. 


432  BACTERIOLOGY. 

claimed  that  a  sort  of  protection  against  certain  specific 
infections  can  also  be  afforded  to  animals  by  the  injection 
into  them  of  cultures  of  entirely  different  species  of 
bacteria,  or  their  products,  and  that  in  some  cases  these 
are  not  of  necessity  of  the  disease-producing  variety. 
For  instance,  Emmerich  and  Mattei1  claim  to  have 
rendered  rabbits  insusceptible  to  anthrax  through  in- 
jections into  them  of  cultures  of  the  streptococcus  of 
erysipelas. 

This,  they  claim,  is  not  due  to  any  antagonism 
between  the  organisms  themselves,  for  in  culture  experi- 
ments the  two  organisms  grew  well  together,  without 
any  alteration  in  their  pathogenic  properties,  but  rather 
to  the  production  of  a  tissue-change  by  which  resistance 
to  the  inroads  of  the  virulent  bacilli  was  established. 
Emmerich  and  Mattei  interpret  this  "  reactive  tissue- 
change  "  as  a  power  acquired  by  the  integral  cells  of  the 
body,  through  the  influence  of  a  stimulus,  of  generating 
a  product  that  is  detrimental  to  the  pathogenic  activity 
of  the  anthrax  bacilli. 

P.awlowsky,2  who  obtained  similar  results  from  the 
introduction  into  the  animal  of  cultures  of  the  bacillus 
prodigiosus,  of  staphylococcus  pyogenes  aureus,  and  of  the 
micrococcus  lanceolatus,  believes  them  to  be  due  to  the 
induction  of  increased  energy  on  the  part  of  the  wander- 
ing cells,  preparing  them  thus  for  the  difficult  task  of 
destroying  the  more  virulent  organisms  with  which  the 
animal  is  subsequently  to  be  inoculated. 

The  experiments  of  G.  and  F.  Klemperer3  upon  acute 
fibrinous  pneumonia,  though  too  limited  in  extent  to 


1  Emmerich  und  Mattei :  Fortschritte  der  Medizin,  1887,  p.  653. 

2  Pawlowski :  Virchow's  Arch.,  vol.  cviii.  p.  494. 

a  G.  and  F.  Klemperer :  Berliner  klin.  Wochenschr.,  1891,  Nos.  34  and  35. 


INFECTION  AND  IMMUNITY.  433 

be  accepted  as  conclusive,  have,  nevertheless,  offered 
a  number  of  most  significant  suggestions,  not  only  in 
connection  with  several  obscure  features  of  this  disease, 
but  also  in  relation  to  the  establishment  of  tissue 
resistance. 

They  found  but  little  difficulty  in  affording  immunity 
to  animals  that  are  otherwise  susceptible  to  the  patho- 
genic action  of  the  organisms  concerned  in  the  pro- 
duction of  this  disease,1  by  the  introduction  into  their 
tissues  of  the  products  of  growth  of  the  organisms  from 
which  the  latter  had  been  separated.  The  immunity 
thus  produced  is  seen  in  some  cases  to  last  as  long  as 
six  months  ;  again  it  is  seen  to  disappear  suddenly  in  a 
way  not  to  be  explained.  It  was  seen  in  one  case  to  be 
hereditary. 

The  energy  of  the  substance  that  has  the  power  of 
affording  immunity  was  seen  to  be  very  much  increased 
by  subjecting  it  to  temperatures  somewhat  higher  than 
that  at  which  it  was  produced  by  the  bacteria.  The 
Klemperers  found  that  if  this  substance  was  heated  to  a 
temperature  of  from  41°  to  42°  C.  for  three  or  four  days, 
or  to  60°  C.  for  from  one  to  two  hours,  intravenous 
injection  was  followed  by  complete  immunity  in  from 
three  to  four  days;  whereas,  if  the  unwarmed  material 
was  used,  immunity  did  not  appear  before  fourteen  days, 
and  then  only  after  the  employment  of  relatively  large 
amounts.  Moreover,  when  the  previously  heated  pro- 
ducts are  introduced  into  the  circulation  of  the  animal, 
the  systemic  reaction  is  of  but  short  duration,  but  if  the 
unwarmed  substance  is  employed,  immunity  is  manifest 

1  Animals  do  not,  as  a  rule,  present  the  pneumonic  changes  seen  in  human 
beings.  The  introduction  of  the  micrococcus  lanceolatus  into  their  tissues 
results,  in  the  case  of  susceptible  animals,  in  the  production  of  septicaemia. 


434  BACTERIOLOGY. 

only  after  the  appearance  of  considerable  elevation  of 
temperature,  which  lasts  for  a  long  time.  In  explana- 
tion of  these  differences,  they  suggest  that,  in  the  latter 
case,  the  high  fever  that  is  seen  to  occur  in  the  animal 
may  serve  to  replace  the  warming  to  which  the  bacterial 
products  had  not  previously  been  subjected,  and  which 
is  necessary  before  they  are  in  a  position  to  bring  about 
the  condition  of  immunity.  They  claim  that  the  bac- 
terial products  employed  in  producing  immunity  in  this 
case  are  not,  in  reality,  the  immunity-affording  substance, 
but  that  they  are  only  the  agents  that  bring  about  in 
the  tissues  of  the  auimal  alterations  that  result  in  the 
production  of  another  body  that  protects  the  animal. 
In  support  of  this,  their  argument  is  that  several  days 
are  necessary  for  the  production  of  immunity  by  the 
introduction  into  the  animal  of  the  bacterial  products ; 
whereas,  if  the  blood-serum  of  this  animal,  which  is 
now  protected,  be  introduced  into  the  circulation  of 
another  animal,  no  such  delay  is  seen,  but  instead,  the 
animal  is  forthwith  protected.  In  the  former  case  the 
actual  protecting  body  had  first  to  be  manufactured  by 
the  tissues ;  whereas,  in  the  second  it  is  already  pre- 
pared, and  is  introduced  as  such  into  the  second  animal. 

They  found  the  serum  of  immunified  animals  to  be  not 
only  capable  of  rendering  other  animals  immune,  but  that 
it  possessed  curative  powers  when  the  disease  is  already 
in  progress.  The  serum  of  immunified  animals,  when 
injected  into  the  circulation  of  animals  in  which  there 
was  a  body- temperature  of  from  40. 4°  to  41°  C.,  reduced 
this  temperature  to  normal  (37.5°  C.)  in  twelve  con- 
secutive experiments  during  the  first  twenty-four  hours 
following  its  employment. 

In  their  opinion,  the  crisis,  seen  in  pneumonia  in 


INFECTION  AND  IMMUNITY.  435 

human  beings,  indicates  the  moment  at  which  the  poison- 
ous products,  manufactured  by  the  bacteria  located  in 
the  lungs,  are  present  in  the  circulation  in  amounts 
sufficient  to  call  forth  in  the  tissues  the  reactive  change 
that  results  in  the  production  of  the  antidotal  substance 
that  has  the  power  of  rendering  the  poisons  inert. 

At  the  time  of  the  crisis  in  pneumonia  the  bacteria 
themselves  are  in  no  way  affected.  They  remain  in 
the  lungs,  and  can  be  detected,  in  full  vigor  and  viru- 
lence, in  the  sputum  of  patients  a  long  time  after  the 
disease  is  cured.  They  have  lost  none  of  their  power 
of  producing  poisonous  products,  and  still  possess  their 
original  pathogenic  relations  toward  susceptible  animals. 
It  is  only  after  the  crisis  that  their  poisons  are  neutral- 
ized by  this  antidotal  proteid  that  has  been  produced 
by  the  cells  of  the  tissues,  and  as  this  occurs  the  sys- 
temic manifestations  gradually  disappear.  The  Klem- 
perers  claim  to  have  isolated  from  cultures  of  the  micro- 
coccus  lanceolatus  a  proteid  body  that  is  the  agent 
concerned  in  producing  the  tissue  reaction  which  results 
in  the  formation  of  the  protecting  substance.  They  like- 
wise isolated  from  the  serum  of  immunified  animals  a 
proteid  that  possesses  the  same  powers  as  the  serum 
itself,  viz.,  of  affording  immunity  and  curing  the  dis- 
ease. 

Here,  again,  it  appears  that  the  processes  of  infection 
and  immunity  are  chemical  in  their  nature,  the  active 
poisons  of  the  invading  organisms — "  the  pneumo- 
toxins " — being  instrumental  in  producing  the  dis- 
eased condition,  while  the  antidotal  or  resisting  body  of 
the  tissues — "  the  anti-pneumotoxin  " — is  the  agent  by 
which  the  poison  is  neutralized. 

Results  in  general  analogous  to  those  of  G.  and  F. 


436  BACTERIOLOGY. 

Klemperer  have  also  been  obtained  by  Emmerich  and 
Fowitzky.1 

In  the  light  of  these  experiments,  the  hypothesis 
advanced  by  Buchner,  that  the  establishment  of  immu- 
nity is  to  be  explained  by  reactive  changes  in  the  in- 
tegral cells  of  the  body,  receives  additional  support, 
and  when  we  consider  the  observations  of  Bitter,2  who 
found  that  in  protective  vaccinations  against  anthrax 
the  vaccines  do  not  disseminate  themselves  through  the 
body,  as  is  the  case  when  the  virulent  organisms  are 
introduced,  but  remain  at  the  point  of  inoculation,  and 
from  this  point  produce,  by  the  absorption  of  their 
chemical  products,  the  systemic  changes  through  which 
the  animal  is  protected  against  subsequent  infection  by 
the  virulent  organisms,  we  feel  justified  in  concluding 
that  the  weight  of  evidence  is  strongly  in  favor  of  this 
view. 

The  experiments  that  have  been  cited  afford  but  an 
imperfect  idea  of  the  enormous  amount  of  work  that  has 
been  done  upon  the  manifold  phases  of  these  important 
subjects ;  they  may,  however,  serve  to  indicate  the  direc- 
tion in  which  the  lines  of  research  have  been  laid.  As 
a  result  of  such  investigations,  our  knowledge  upon  in- 
fection and  immunity  may  at  present  be  summarized 
about  as  follows : 

1.  That  infection  may  be  considered  as  a  contest 
between  bacteria  and  living  tissues,  conducted  on  the 
part  of  the  former  by  means  of  the  poisonous  products 
of  their  growth,  and  resisted  by  the  latter  through  the 
agency  of  proteid  bodies  normally  present  in  and  gener- 
ated by  their  integral  cells. 

1  Emmerich  and  Fowitzky  ;  Munchener  med.  Wochenschr.,  1891,  No.  32. 

2  Bitter :  Zeitschrift  fur  Hygiene,  1888,  Bd.  iv. 


INFECTION  AND  IMMUNITY.  437 

2.  That  when  infection  occurs  it  may  be  explained 
either  by  the  excess  of  vigor  of  the  bacterial  products 
over  the  antidotal  or  protective  proteids  produced  by 
the  tissues,  or  to  some  cause  that  has  interfered  with 
the  normal  activity  and  production  of  these  bodies. 

3.  That  immunity  is  most  frequently  seen  to  follow 
the  introduction  into  the  body  of  the  products  of  growth 
of  bacteria  that  in  some  way  or  other  have  been  mod- 
ified.    This  modification  may  be  artificially  produced  in 
the  products  themselves  of  virulent  organisms,  and  then 
introduced  into  the  tissues  of  the  auimal ;   or  the  viru- 
lent bacteria  may  be  so  treated  that  they  are  no  longer 
of  full  virulence,  and  when  introduced  into  the  body 
of  the  auimal  will   produce  poisons  of  a   much   less 
vigorous  nature  than  would  otherwise  be  the  case. 

4.  That    immunity   following    the    introduction    of 
bacterial  products  into  the  tissues  is  not  in  all  cases  the 
result  of  the  permanent  presence  of  these  substances, 
per  se,  in  the  tissues,  or  of  a  tolerance  acquired  by  the 
tissues  to  them,  but  is  probably,  in  certain  instances,  due 
to  the  formation  in  the  tissues  of  another  body  that  acts 
as  a  protecting  antidote  to  the  poisonous  products  of 
invading  organisms. 

5.  That  this  protecting  proteid  that  is  generated  by 
the  cells  of  the  tissues  need  not  of  necessity  be  antago- 
nistic to  the  life  of  the  invading  organisms  themselves, 
but  in  some  cases  must  be  looked  upon  more  as  an  anti- 
dote to  their  poisonous  products. 

6.  That  in  the  serum  of  the  normal  circulating  blood 
of  many  animals  there  exists  a  substance  that  is  capable, 
outside  of  the  body,  of  rendering  inert  bacteria  that,  if 
introduced  into  the  body  of  the  auimal,  would  prove 
infective. 


438  BACTERIOLOGY. 

7.  That  phagocytosis,  though  frequently  observed,  is 
not  essential  to  the  establishment  of  immunity,  but  is 
more  probably  a  secondary  process,  the  bacteria  being 
taken   up  by  the  leucocytes   only  after   having   been 
modified  in  virulence  through  the  normal  germicidal 
activity  of  the  serum  of  the  blood  and  of  other  fluids  in 
the  body. 

8.  That,  of  the  hypotheses  advanced  in  explanation 
of  acquired  immunity,  the  one  worthy  of  greatest  con- 
fidence is  that  which  assumes  immunity  to  be  due  to 
reactive  changes  on  the  part  of  the  tissues  that  result  in 
the  formation  in  these  tissues  of  antitoxic  substances 
capable  of  neutralizing  the  poisons    produced  by  the 
bacteria  against  which  the  animal  has  been  immunified. 


CHAPTER  XXVII. 

Bacteriological  study  of  water— Methods  employed— Precautions  to  be 
observed— Apparatus  used,  and  methods  of  using  them— Methods  of  investi- 
gating air  and  soil. 

THE  conditions  that  favor  the  epidemic  outbreak  of 
typhoid  fever,  Asiatic  cholera,  and  other  maladies  of 
which  these  may  be  taken  as  types,  have  served  as  a 
subject  for  discussion  by  sanitarians  for  a  long  time. 

Of  the  hypotheses  that  have  been  advanced  in  explan- 
ation of  the  existence  and  dissemination  of  these  dis- 
eases, two  stand  pre-eminent  and  are  worthy  of  con- 
sideration. They  are  the  u  ground  -water "  theory  of 
von  Pettenkofer  and  his  pupils,  and  the  "drinking- 
water"  theory  of  the  school  of  bacteriologists  of  which 
Koch  stands  at  the  head. 

The  adherents  to  the  "ground -water7'  view  explain 
the  presence  of  these  diseases  in  epidemic  form  through 
alterations  in  the  soil  resulting  from  fluctuations  in  the 
level  of  the  soil  water,  and  assign  to  the  drinking-water 
either  a  very  insignificant  rdle,  or,  as  is  most  frequently 
the  case,  ignore  it  entirely.  On  the  other  hand,  those 
who  have  been  instrumental  in  developing  the  drinking- 
water  hypothesis,  claim  that  alterations  in  the  soil  play 
little  or  no  part  in  favoring  the  appearance  of  these 
diseases  in  a  neighborhood,  but  that,  as  a  rule,  they 
appear  as  a  result  of  direct  infection,  through  the  use  of 
waters  that  are  contaminated  with  materials  containing 


440  BACTERIOLOGY. 

the  specific  organisms  that  are  known  to  cause  such 
diseases. 

As  a  result  of  many  observations  ori  both  sides  of  the 
question,  the  evidence  is  greatly  in  favor  of  the  opinion 
that  polluted  water  is  primarily  the  underlying  cause  of 
these  epidemics,  and  this  too,  very  often,  when  the  state 
of  the  soil  water,  in  the  light  of  the  "  ground- water " 
hypothesis,  is  just  the  reverse  of  what  it  should  be  in 
order  to  render  it  answerable  for  them.  It  is  manifest, 
therefore,  that  the  careful  bacteriological  study  of  water 
intended  for  domestic  use  is  of  the  greatest  importance, 
and  should  be  a  routine  procedure  in  all  communities 
receiving  their  water  supply  from  sources  that  are  liable 
to  pollution. 

The  object  aimed  at  in  such  investigations  should  be 
to  determine  if  the  water  approaches  constancy  in  the 
number  and  kind  of  bacteria  contained  in  it — for  all 
waters,  except  deep  ground  water,  contain  bacteria ;  if 
sudden  fluctuations  in  the  number  of  bacteria  occur  in 
these  waters,  and  if  so,  to  what  they  are  due;  and  finally, 
and  most  important,  does  the  water  contain  constantly, 
or  at  irregular  periods,  bacteria  that  can  be  traced  to 
human  excrement,  not  of  necessity  pathogenic  varieties, 
but  bacteria  that  are  known  to  be  present  normally  in 
the  intestinal  canal?  For,  if  conditions  are  favorable  to 
,the  presence  of  these  varieties  the  same  conditions  would 
favor  the  admission  to  the  water  of  other  forms  of  bac- 
teria that  are  concerned  in  the  production  of  diseases  in 
the  intestines. 

In  considering  water  from  a  bacteriological  stand- 
point, it  must  always  be  borne  in  mind  that  comparisons 
with  any  general  fixed  standard  are  not  of  much  value, 
for  just  as  normal  waters  from  different  sources  are  seen 


BACTERIOLOGICAL  STUDY  OF  WATER.       441 

to  present  variations  in  their  chemical  composition,  with- 
out being  unfit  for  use,  so  may  the  number  of  bacteria 
per  volume  in  water  from  one  source  be  always  greater 
or  smaller  than  in  that  from  another,  and  yet  no  differ- 
ence may  be  seen  to  result  from  their  employment.  For 
this  reason  the  proper  study  of  any  water,  from  this 
point  of  view,  should  begin  with  the  establishment  of 
what  may  be  called  its  normal  mean  number  of  bacteria, 
as  well  as  the  character  of  the  prevailing  species;  and 
in  order  to  do  this  the  investigations  must  cover  a  long 
period  of  time  through  all  the  seasonal  variations  of 
weather.  From  data  obtained  in  this  way  it  may  be 
possible  to  predict  approximately  the  normal  bacterio- 
logical condition  of  water  at  any  season.  Marked  de- 
viations from  these  "  means,"  either  in  the  quantity  or 
quality  of  the  organisms  present,  can  then  be  considered 
as  indicative  of  the  existence  of  some  unusual,  disturbing 
element,  the  nature  of  which  should  be  investigated. 
Similarly,  it  is  impossible  to  formulate  an  opinion  of 
much  value  from  a  single  chemical  analysis  of  a  water, 
for  the  results  thus  obtained  indicate  only  the  state  of 
the  water  at  the  time  the  sample  was  procured,  and  give 
no  indication  as  to  whether  it  differed  at  that  time  from 
its  usual  condition,  or  from  the  normal  condition  of  the 
water  throughout  the  immediate  neighborhood. 

The  interpretation  of  the  results  of  both  chemical  and 
bacteriological  analysis  of  a  sample  of  water  acquires  its 
full  value  only  through  comparison,  either  with  "  means'7 
that  have  been  determined  for  this  water,  or  with  the 
results  of  simultaneous  analyses  of  a  number  of  samples 
from  the  other  sources  of  supply  of  the  locality. 

The  aid  of  the  bacteriologist  is  frequently  sought  in 
connection  with  investigations  upon  waters  that  are 


442  BACTERIOLOGY. 

supposed  to  be  concerned  in  the  production  of  disease, 
particularly  typhoid  fever,  either  in  isolated  cases  or  in 
widespread  epidemic  outbreaks,  and  almost  as  often  do 
reliable  bacteriologists  fail  to  detect  the  bacillus  that  is 
the  cause  of  typhoid  fever  in  these  waters. 

The  failure  to  find  the  organisms  of  typhoid  fever  in 
water  by  the  usual  methods  of  analysis  does  not  by  any 
means  prove  that  they  are  not  present  or  have  not  been 
present.  The  means  that  are  ordinarily  employed  in 
the  work  admit  of  such  a  very  small  volume  of  water 
being  used  in  the  test  that  we  can  readily  understand  how 
these  organisms  might  be  present  in  moderate  numbers 
and  yet  none  of  them  be  included  in  the  drop  or  two 
of  the  water  that  are  taken  for  study.  The  conditions 
are  not  those  of  a  solution,  each  drop  of  which  contains 
exactly  as  much  of  the  dissolved  material  as  do  all  other 
drops  of  equal  volume,  but  are  rather  those  of  a  suspen- 
sion in  each  drop  or  volume  of  which  the  number  of 
suspended  particles  are  liable  to  the  greatest  degree  of 
variation.  Furthermore,  there  are  other  reasons  that 
would,  a  priori,  preclude  our  expecting  to  find  the 
typhoid  bacilli  in  water  in  which  we  may  have  reason 
to  believe  they  had  been  deposited,  viz.,  attention  is  not 
usually  directed  to  the  water  until  the  presence  of  the 
disease  has  become  conspicuous,  usually  in  from  three 
to  four  weeks  after  the  time  when  the  pollution  probably 
occurred.  Three  or  four  weeks  is  ordinarily  sufficient 
time  for  the  delicate,  non-resistant  bacillus  of  typhoid 
fever  to  succumb  to  the  unfavorable  conditions  under 
which  it  finds  itself  in  water.  By  unfavorable  condi- 
tions is  meant  the  absence  of  suitable  nutrition ;  unfavor- 
able temperature ;  probably  the  antagonistic  influence  of 
more  hardy  saprophytic  bacteria,  particularly  the  so- 


BACTERIOLOGICAL  STUDY  OF  WATER        443 

called  "  water  bacteria,"  and  of  more  highly  organized 
water  plants ;  the  effect  of  mechanical  precipitation ;  and 
of  great  importance,  the  disinfecting  action  of  direct 
sunlight. 

Though  it  is  so  rare  as  to  be  almost  never,  that 
typhoid  bacilli  are  found  in  drinking-water,  it  must, 
nevertheless,  not  be  supposed  that  bacteriological  anal- 
yses of  suspicious  waters  shed  no  light  upon  the  exist- 
ence of  pollution  and  the  suitability  or  non-suitability 
of  the  water  for  drinking  purposes. 

In  the  normal  intestinal  tract  of  all  human  beings, 
and  many  other  mammals,  as  well  as  associated  with  the 
specific  disease-producing  bacterium  in  the  intestines  of 
typhoid-fever  patients,  is  an  organism  that  is  frequently 
found  in  polluted  drinking-waters,  and  whose  presence 
is  proof  positive  of  pollution  by  either  normal  or  dis- 
eased intestinal  contents ;  and  though  efforts  may  result 
in  failure  to  detect  the  specific  bacillus  of  typhoid  fever, 
the  finding  of  the  other  organism,  the  bacterium  coli  com- 
mune, justifies  one  in  expressing  the  opinion  that  the 
water  under  consideration  has  been  polluted  by  intes- 
tinal evacuations  from  either  human  beings  or  animals. 
Waters  so  located  as  to  be  liable  to  such  pollution  can 
never  be  considered  as  other  than  a  continuous  source  of 
danger  to  those  using  them. 

Another  point  to  be  remembered  is  in  connection  with 
the  value  of  chlorine  as  indicative  of  contamination  by 
human  excrement.  It  is  commonly  taught  that  an  ex- 
cessive amount  of  chlorine  in  water  points  to  contamina- 
tion by  human  excreta.  This  may  or  may  not  be  true 
according  to  circumstances.  A  high  proportion  of  this 
substance  in  a  sample  of  water  from  a  locality,  the  neigh- 
boring waters  of  which  are  poor  in  chlorine,  is  unques- 


444  BACTERIOLOGY. 

tionably  a  suspicious  indication,  but  in  a  district  close 
to  the  sea  or  near  salt  deposits,  for  instance,  where  the 
water  generally  is  high  in  chlorine,  the  value  of  the 
indications  thus  afforded  is  very  much  diminished  unless 
the  amount  found  in  the  sample  under  examination 
greatly  exceeds  the  normal  "  mean/7  previously  deter- 
mined, for  the  amount  of  chlorine  in  the  waters  of  the 
neighborhood. 

A  striking  example  of  such  a  condition  as  the  latter 
recently  occurred  in  the  experience  of  the  writer 'while 
inspecting  a  group  of  water  supplies  on  the  east  coast  of 
Florida.  In  each  instance  the  water  was  obtained  from 
properly  protected  artesian  wells,  ranging  from  200  to 
400  feet  deep,  and  located  within  a  few  hundred  yards 
of  the  sea.  The  first  sample  that  was  subjected  to  chem- 
ical analysis  revealed  such  an  unusually  high  proportion 
of  chlorine  that,  had  this  sample  alone  been  considered, 
the  opinion  that  it  was  polluted  by  human  excreta  might 
have  been  advanced.  To  prevent  such  an  error  samples 
of  water  from  a  number  of  wells  in  the  neighborhood 
were  examined,  and  they  were  all  found  to  contain  from 
ten  to  twelve  times  the  amount  of  chlorine  that  ordi- 
narily appears  in  inland  waters,  the  excess  being  evi- 
dently due  to  leakage  through  the  soil  into  the  wells  of 
water  from  the  sea.  In  short,  the  presence  of  an  excess  of 
chlorine  in  water,  while  often  indicating  pollution  from 
human  evacuations,  may,  nevertheless,  sometimes  arise 
from  other  sources,  but  the  presence  in  water  of  bacteria 
normally  found  in  the  intestinal  canal  can  manifestly 
admit  of  but  one  interpretation,  viz.,  that  fecal  matters 
have  at  some  time  and  place  been  deposited  in  this 
water,  and  that  while  no  specific  disease-producing  or- 
ganisms may  have  been  detected,  still,  waters  in  which 


BACTERIOLOGICAL  STUDY  OF  WATER.       445 

such  pollutions  are  possible  are  a  constant  menace  to 
the  health  of  those  who  use  them  for  domestic  purposes. 

A  sudden  variation  from  the  normal,  mean  number 
of  bacteria,  or  from  the  normal  chemical  composition  of 
a  water,  calls  at  once  for  a  thorough  inspection  of  the 
supply,  while  at  the  same  time  the  characters  of  the 
organisms  present  are  to  be  subjected  to  the  most  care- 
ful study. 

THE  QUALITATIVE  BACTERIOLOGICAL  ANALYSIS 
OF  WATER. — The  qualitative  bacteriological  analysis 
of  water  entails  much  labor,  as  it  requires  not  only  that 
all  the  different  species  of  organism  found  in  the  water 
should  be  isolated,  but  that  each  representative  should 
be  subjected  to  systematic  study,  and  its  pathogenic  or 
non- pathogenic  properties  determined. 

For  this  purpose  the  methods  for  the  isolation  of 
individual  species  which  have  already  been  described, 
and  the  means  of  studying  these  species  when  isolated, 
are  indispensable. 

For  this  analysis  certain  precautions  essential  to  ac- 
curacy are  always  to  be  observed. 

The  sample  is  to  be  collected  under  the  most  rigid 
precautions  that  will  exclude  organisms  from  sources 
other  than  that  under  consideration.  If  drawn  from  a 
spigot,  it  should  never  be  collected  until  the  water  has 
been  flowing  for  fifteen  to  twenty  minutes  in  a  full 
stream.  If  obtained  from  a  stream  or  a  spring,  it 
should  be  collected,  not  from  the  surface,  but  rather 
from  about  one  foot  beneath  the  surface. 

It  should  always  be  collected  in  vessels  which  have 
previously  been  thoroughly  freed  from  all  dirt  and 
organic  particles,  and  then  sterilized.  And>  the  plates 


20 


446  BACTERIOLOGY. 

should  be  made  as  quickly  as  possible  after  collecting 
the  sample. 

When  circumstances  permit,  all  water  analyses  should 
be  made  on  the  spot  at  which  the  sample  is  taken,  as  it 
is  known  that  during  transportation,  unless  the  samples 
are  kept  packed  in  ice,  a  multiplication  of  the  organisms 
contained  in  it  always  occurs. 

For  the  purpose  of  qualitative  analysis  it  is  necessary 
that  a  small  portion  of  the  water — one,  two,  three,  five 
drops — should  first  be  employed  as  the  amounts  from 
which  plates  are  to  be  made.  In  this  way  one  forms 
some  idea  as  to  the  approximate  number  of  organisms 
in  the  water,  and,  can,  in  consequence,  determine  the 
amount  of  water  necessary  to  use  for  each  set  of  plates. 
Duplicate  plates  are  always  to  be  made — one  set  upon 
agar-agar,  which  are  to  be  kept  in  the  incubator  at  body 
temperature,  and  one  set  upon  gelatin,  to  be  kept  at 
from  18°  to  20°  C. 

As  soon  as  the  colonies  have  developed  the  plates  are 
to  be  carefully  compared  and  studied.  It  is  to  be  noted 
if  any  difference  in  the  appearance  of  the  organisms  on 
corresponding  plates  exists,  and  if  so,  to  what  is  it  due  ? 
It  is  to  be  particularly  noted  which  plates  contain  the 
greater  number  of  colonies,  those  kept  at  the  higher  or 
those  at  the  lower  temperature.  In  this  way  the  tem- 
perature best  suited  for  the  growth  of  the  majority  of 
these  organisms  may  be  determined. 

As  a  rule,  the  greater  number  of  colonies  appears 
upon  the  gelatin  plates  that  are  kept  at  18°  to  20°  C., 
and  from  this  it  would  seem  that  many  of  the  normal 
water  bacteria  do  not  find  the  higher  temperature  so 
favorable  to  their  development  as  do  the  organisms  not 


BACTERIOLOGICAL  STUDY  OF  WATER.       447 

naturally  present  in   water,  particularly  the  pathogenic 
varieties. 

NOTE. — In  determining  if  the  organisms  found  are 
possessed  of  pathogenic  properties,  in  what  way  will 
your  tests  be  influenced  by  this  observation  ? 

From  recent  investigations  upon  this  subject  it  ap- 
pears that  the  difference  in  behavior  toward  heat  of 
bacteria  present  in  water  may  have  a  very  important 
application.  Dr.  Theobald  Smith  has  recently  suggested 
a  method  by  which  it  is  easily  possible  to  isolate,  from 
waters  in  which  they  are  present,  certain  organisms  that 
are  of  the  utmost  importance  in  influencing  our  judg- 
ment upon  the  fitness  of  the  water  for  domestic  use. 
By  the  addition  of  small  quantities,  one,  two,  or  three 
drops  of  the  suspicious  water  to  fermentation  tubes  (see 
article  on  Fermentation  Tube)  containing  bouillon  to 
which  2  per  cent,  of  glucose  has  been  added,  and  keep- 
ing them  at  the  temperature  of  the  body,  37°  to  38° 
C.,  the  growth  of  the  intestinal  bacteria  that  may  be 
present  in  the  water  is  favored,  while  that  of  the  water 
organisms  is  not ;  in  consequence,  after  from  thirty-six 
to  forty-eight  hours  the  fermentation,  characteristic  of 
most  of  these  organisms,  is  evidenced  by  the  accumula- 
tion of  gas  in  the  closed  end  of  the  tube.  From  these 
tubes  the  growing  bacteria  can  then  be  easily  isolated 
by  the  plate  method,  and  it  will  not  be  infrequent  to 
find  intestinal  bacteria  present  in  pure  culture. 

Another  method  for  the  same  object  is  to  collect  a 
sample  of  about  100  c.c.  of  the  water  to  be  tested  in  a 
sterilized  flask,  and  add  to  this  about  25  c.c.  of  steril- 
ized bouillon  of  four  times  the  usual  strength.  This 


448  BACTERIOLOGY. 

is  then  placed  in  the  incubator  at  37°  to  38°  C.,  for 
thirty-six  to  forty-eight  hours,  after  which  plates  are 
to  be  made  from  it  in  the  usual  way  ;  the  results  will 
often  be  a  pure  culture  of  some  single  organism,  either 
one  of  the  intestinal  variety  or  a  closely  allied  species. 
By  a  method  analogous  to  the  latter  the  spirillum  of 
Asiatic  cholera  has  been  isolated  from  water ;  and  by 
taking  advantage  of  the  effect  of  elevated  temperature 
upon  the  bacteria  of  water,  Dr.  Yaughan,  of  Michigan, 
has  succeeded  in  isolating  from  suspicious  waters  a 
group  of  organisms  very  closely  allied  to  the  bacillus 
of  typhoid  fever. 

THE  QUANTITATIVE  ESTIMATION  OF  BACTEKIA  IN 
WATER. — Quantitative  analysis  requires  more  care  in 
the  measurement  of  the  exact  volume  of  water  employed, 
for  the  results  are  to  be  expressed  in  terms  of  the  num- 
ber of  individual  organisms  to  a  definite  volume.  The 
necessity  for  making  the  plates  at  the  place  at  which  the 
sample  is  collected  is  to  be  particularly  accentuated  in 
this  analysis,  for  the  multiplication  of  the  organisms 
during  transit  is  so  great  that  the  results  of  analyses 
made  after  the  water  has  been  in  a  vessel  for  a  day  or 
two  are  often  very  different  from  those  that  would  have 
been  obtained  on  the  spot. 

NOTE. — Inoculate  a  tube  containing  about  ten  cubic 
centimetres  of  sterilized  distilled  or  tap  water  with  a 
very  small  quantity  of  a  solid  culture  of  some  one  of 
the  organisms  with  which  you  have  been  working, 
taking  care  that  none  of  the  culture  medium  is  intro- 
duced into  the  water-tube  and  that  the  bacteria  are 
evenly  distributed  through  it.  Make  plates  at  once, 
and  on  each  succeeding  day,  from  this  tube,  and  deter- 


BACTERIOLOGICAL  STUDY  OF  WATER.       449 

mine  by  counts  whether  there  is  an  increase  or  diminu- 
tion in  the  number  of  organisms — i.  e.,  if  they  are  grow- 
ing or  dying.  Represent  the  results  graphically,  and  it 
will  be  noticed  that  in  many  cases  there  is  at  first,  during 
the  first  three  or  four  days,  a  multiplication,  after  which 
there  is  a  rapid  diminution  ;  and,  if  the  organism  does 
not  form  spores,  usually  complete  death  in  from  ten  to 
twelve  days.  This  is  not  true  for  all  organisms,  but 
does  hold  for  many. 

Where  it  is  not  convenient,  however,  to  make  the 
analysis  on  the  spot,  the  sample  of  water  should  be  col- 
lected and  packed  in  ice  and  kept  on  ice  until  ready  for 
use,  which  should  in  all  cases  be  as  soon  after  its  collec- 
tion as  possible. 

For  the  collection  of  water  for  this  purpose,  a  con- 
venient vessel  to  be  employed  is  a  glass  bulb  (Fig.  91) 
or  balloon,  which  one  soon  learns  to  make  for  oneself 
from  glass  tubing. 

FIG.  91. 


g 
Glass  bulb  for  collecting  samples  of  water. 

It  consists  simply  of  a  round  glass  sphere  blown  on 
the  end  of  a  glass  tube,  which  latter  is  subsequently 
drawn  out  into  a  fine  capillary  stem  and  sealed  while 
hot.  As  it  cools,  the  contraction  of  the  air  within  the 
bulb  results  "in  the  production  of  a  negative  pressure. 
If  the  point  of  the  stem  be  broken  off  under  water,  the 
water  is  pressed  up  into  the  bulb,  because  of  the  exist- 
ence of  the  negative  pressure  within.  The  negative 


450  BACTERIOLOGY. 

pressure  obtained  in  this  way  is  frequently  not  sufficient 
to  permit  of  the  bulb  being  completely  filled,  and  often 
only  a  few  drops  of  fluid  can  be  obtained.  To  obviate 
this  the  bulbs  may  be  blown  and  allowed  to  cool,  but 
not  sealed.  After  a  sufficient  number  of  them  are  pre- 
pared they  are  taken,  one  at  a  time,  and  gently  warmed 
over  the  flame ;  while  still  warm  the  extremity  of  the 
stem  is  dipped  into  distilled  water  and  held  there  until 
a  few  drops  have  passed  up  into  the  bulb ;  this  is  then 
carefully  boiled,  or  rather,  completely  vaporized,  over  the 
flame,  and  while  the  steam  is  still  escaping  the  point  is 
sealed  in  the  gas  flame.  All  air  will  have  thus  been 
replaced  by  water  vapor,  and  if  the  point  of  the  stem 
be  now  broken  off  under  water  the  bulb  will  fill  quickly 
and  completely.  It  is  not  desirable  to  fill  them  com- 
pletely, but  rather  to  only  about  three-fourths  of  their 
capacity,  as  when  full  it  is  difficult  to  empty  them  with- 
out contaminating  the  contents.  They  are  emptied  by 
gently  warming  over  a  gas  or  alcohol  flame. 

A  number  of  them  may  be  made,  sealed,  and  kept  on 
hand.  They  are  sterile  so  long  as  they  are  sealed,  be- 
cause of  the  heat  that  is  employed  in  their  manufacture. 

When  a  sample  of  water  is  to  be  taken,  the  point  of 
a  bulb  is  simply  broken  off  with  sterilized  forceps  under 
water  at  the  place  from  which  the  sample  is  to  be  pro- 
cured and  held  there  until  the  necessary  amount  has 
been  obtained.  This  may  serve  as  a  sample  from  which 
to  prepare  plates  or  Esmarch  tubes  on  the  spot,  or  the 
tip  of  the  stem  may  be  resealed  in  the  flame  of  an  alco- 
hol lamp,  the  bulb  packed  in  ice,  and  transported  in  this 
condition  to  the  laboratory. 

Another  very  simple  and  useful  device  for  collecting 
water  samples  is  that  recommeded  by  Kirschner.  It 


BACTERIOLOGICAL  STUDY  OF  WATER.       451 

consists  of  a  piece  of  glass  tubing  of  about  5  or  6  mm. 
inside  diameter  and  36  cm.  long,  bent  in  the  form  of  a 
U,  with  either  extremity  of  the  arms  bent  again  at  right 
angles  in  the  same  plane  and  drawn  out  to  a  point  and 
sealed.  They  are  sterilized  in  the  flame  as  they  are 
made.  The  sample  is  collected  by  breaking  off  both 
points,  immersing  one  of  them  in  the  water  and  suck- 
ing on  the  other  until  the  tube  is  filled.  Then  both 
points  are  again  sealed  in  the  flame  and  the  tube  packed 
in  ice.  The  objection  to  this  tube  is  the  danger  of  con- 
taminating its  contents  with  saliva  during  the  act  of  fill- 
ing by  suction,  though  this  danger  is  not  so  great  as 
might  at  first  appear,  as  we  shall  learn  in  our  efforts  to 
cultivate  bacteria  from  the  mouth  cavity. 

NOTE. — Make  cover-slips  from  your  own  mouth  ; 
make  plates  on  both  gelatin  and  agar-agar,  at  the  same 
time.  Compare  the  number  of  bacteria  found  by  micro- 
scopic examination  of  the  cover-slips  with  the  number 
of  colonies  that  develop  on  the  plates. 

In  beginning  the  quantitative  analysis  of  water  with 
which  one  is  not  acquainted,  there  are  certain  prelimi- 
nary steps  that  are  essential. 

It  is  necessary  to  know  approximately  the  number  of 
organisms  contained  in  any  fixed  volume,  so  as  to  deter- 
mine the  quantity  of  water  to  be  employed  for  the  plates 
or  tubes.  This  is  usually  done  by  making  preliminary 
plates  from  one  drop,  two  drops,  0.25  c.c.,  0.5  c.c.,  and 
1  c.c.  of  the  water.  After  each  plate  has  been  labelled 
with  the  amount  of  water  used  in  making  it,  it  is  placed 
aside  for  development.  When  this  has  occurred,  one 
selects  the  plate  upon  which  the  colonies  are  only  mod- 


452  BACTERIOLOGY. 

erate  in  number — about  200  to  300  colonies  presenting 
— and  employs  in  the  subsequent  analysis  the  same 
amount  of  water  that  was  used  in  making  this  plate. 

If  the  original  water  contained  so  many  organisms 
that  there  developed  on  a  plate  or  tube  made  with  one 
drop  too  many  colonies  to  be  easily  counted,  then  the 
sample  must  be  diluted  with  one,  ten,  twenty-five,  fifty, 
or  one  hundred  volumes,  as  the  case  may  require,  of 
sterilized  distilled  water.  •  This  dilution  must  be  accu- 
rate, and  its  exact  extent  noted,  so  that  subsequently  the 
number  of  organisms  per  volume  in  the  original  water 
may  be  calculated. 

The  use  of  a  drop  is  not  sufficiently  accurate.  The 
dilution  should  therefore  always  be  to  a  degree  that  will 
admit  of  the  employment  of  a  volume  of  water  that  may 
be  exactly  measured,  0.25,  0.5  c.c.  being  the  amounts 
most  convenient  for  use. 

Duplicate  plates  should  always  be  made  and  the  mean 
of  the  number  of  colonies  that  develop  upon  them  taken 
as  the  basis  from  which  to  calculate  the  number  of  or- 
ganisms per  volume  in  the  original  water. 

For  example:  From  a  sample  of  water,  0.25  c.c.  is 
added  to  a  tube  of  liquefied  gelatin,  carefully  mixed  and 
poured  out  as  a  plate.  When  development  occurs,  the 
number  of  colonies  are  too  numerous  to  be  accurately 
counted.  One  cubic  centimetre  of  the  original  water  is 
then  to  have  added  to  it,  under  precautions  that  pre- 
vent contamination  from  without,  99  c.c.  of  sterilized 
distilled  water — that  is,  we  have  now  a  dilution  of 
1  : 100.  Again,  0.25  c.c.  of  this  dilution  is  plated  and 
we  find  180  colonies  on  the  plate.  Assuming  that  each 
colony  develops  from  an  individual  bacterium,  though 
this  is  perhaps  not  strictly  true,  we  had  180  organisms 


BACTERIOLOGICAL  STUDY  OF  WATER.       453 

in  0.25  c.c.  of  our  1  : 100  dilution,  therefore  in  0.25  c.c. 
of  the  original  water  we  had  180  X  100  =  18,000  bac- 
teria, which  will  be  72,000  bacteria  per  cubic  centimetre 
(0.25  =  18,000,  1  c.c.  =  18,000  X  4  =  72,000).  The  re- 
sults are  always  to  be  expressed  in  terms  of  the  number 
of  bacteria  per  cubic  centimetre  of  the  original  water. 

Another  point  of  very  great  importance  (already 
mentioned)  is  the  effect  of  temperature  upon  the  num- 
ber of  colonies  of  bacteria  that  will  develop  on  plates 
made  from  water.  It  must  always  be  remembered 
that  a  larger  number  of  colonies  appear  on  gelatin 
plates  made  from  water  and  kept  at  18°  to  20°  C.  than 
on  agar-agar  plates  kept  in  the  incubator.  The  follow- 
ing table,  illustrative  of  this  point,  gives  the  results  of 
parallel  analyses  of  the  same  waters,  the  one  series  of 
counts  having  been  made  upon  gelatin  plates  at  the 
ordinary  temperature  of  the  room,  the  other  upon  plates 
of  agar-agar  kept  for  the  same  length  of  time  in  the 
incubator  at  from  37°  to  38°  C.  It  will  be  seen  from 
the  table  that  much  the  larger  number  of  colonies,  i.  e., 
much  higher  results,  are  always  obtained  when  gelatin 
is  employed.  The  importance  of  this  point  in  the 
quantitative  bacteriological  analysis  of  water  is  too 
apparent  to  require  further  comment. 


20* 


454  BACTERIOLOGY. 


TABLE  ILLUSTRATING  THE  PROPORTION  BETWEEN  THE  RE- 
SULTS OBTAINED  BY  THE  USE  OF  GELATIN  AND  AGAR-AGAR 
IN  QUANTITATIVE  BACTERIOLOGICAL  ANALYSIS  OF  WATER. 
RESULTS  RECORDED  ARE  THE  NUMBER  OF  COLONIES  THAT 
DEVELOPED  FROM  THE  SAME  AMOUNT  OF  WATER  IN  EACH 
SERIES.* 

NUMBER  OF  COLONIES  FROM  WATER  THAT  DEVELOPED  UPON— 

Gelatin  plates  at  18°  to  20°  C.               Agar-agar  plates  at  37°  to  38°  C. 

310  ........        170 

280  ...>....        140 

310)  fl80 

340  *  1 160 

6501  f210 

630  F  1 320 

380  )  f  290 

400)  '  1 210 

10001  (100 

890)  1 130 

340!  f280 

370  <  1210 

490  )  f 110 

580  S  1 100 

Throughout  this  part  of  the  work  it  is  to  be  borne 
in  mind  that  when  one  refers  to  plates  it  is  not  to  a 
set,  as  in  the  isolation  experiments,  but  to  a  single 
plate. 

METHOD  OF  COUNTING  THE  COLONIES  ON  PLATES. 
— For  convenience  in  counting  colonies  on  plates  or  in 
tubes,  it  is  customary  to  divide  the  whole  area  of  the 
gelatin  occupied  by  colonies  into  smaller  areas,  and 
either  count  all  the  colonies  in  each  of  these  areas  and 
add  the  several  sums  together  for  the  total ;  or  to  count 
the  number  of  colonies  in  each  of  several  areas,  ten  or 
twelve,  take  the  mean  of  the  results  and  multiply  this 
by  the  number  of  areas  containing  colonies. 

1  I  am  indebted  to  Dr.  James  Homer  Wright,  Thomas  Scott  Fellow  in 
Hygiene  (1892-93),  University  of  Pennsylvania,  for  the  results  presented  in 
this  table. 


BACTERIOLOGICAL  STUDY  OF  WATER.       455 

By  this  latter  method,  however,  the  results  vary  so 
much  in  different  counts  of  the  same  plate  that  they 
cannot  be  considered  as  more  than  rough  approxima- 
tions. 

NOTE. — Prepare  a  plate;  calculate  the  number  of 
colonies  upon  it  by  this  latter  method.  Now  repeat 
the  calculation,  making  the  average  from  another  set  of 
squares.  Now  actually  count  the  entire  number  of 
colonies  on  the  plate.  Compare  the  results. 

For  facilitating  the  counting  of  colonies  several  very 
convenient  devices  exist. 

WOLFFHUGEL'S  COUNTING  APPARATUS. — This  ap- 
paratus (Fig.  92)  consists  of  a  flat  wooden  stand,  the 
centre  of  which  is  cut  out  in  such  a  way  that  either'a 

PIG.  92. 


WolffhiigeFs  apparatus  for  counting  colonies. 

black  or  white  glass  plate  may  be  placed  in  it.  These 
form  a  background  upon  which  the  colonies  may  more 
easily  be  seen  when  the  plate  to  be  counted  is  placed 


456  BACTERIOLOGY. 

upon  it.  When  the  gelatin  plate  containing  the  colo- 
nies has  been  placed  upon  this  background  of  glass, 
it  is  then  covered  by  a  transparent  glass  plate  which 
swings  on  a  hinge.  This  plate,  which  is  ruled  in  square 
centimetres  and  subdivisions,  when  in  position,  is  just 
above  the  colonies  without  touching  them. 

The  gelatin  plate  is  moved  about  until  it  rests  under 
the  centre  of  the  area  occupied  by  the  ruled  lines. 

The  number  of  colonies  in  each  square  centimetre  is 
then  counted,  and  the  sum -total  of  the  colonies  in  all 
these  areas  gives  the  number  of  colonies  on  the  plate ; 
or,  as  has  already  been  indicated,  if  the  number  of  col- 
onies be  very  great  a  mean  may  be  taken  of  the  number 
in  several  (6  or  8)  squares ;  this  is  to  be  multiplied  by 
the  total  number  of  squares  occupied  by  the  gelatin. 
The  result  is  an  approximation  of  the  total  number  of 
colonies. 

When  the  colonies  are  quite  small,  as  is  frequently 
the  case,  the  counting  may  be  rendered  easier  by  the 
use  of  a  small  hand-lens. 

FIG.  93. 


Lens  for  counting  colonies. 

In  Fig.  93  is  seen  the  form  of  hand-lens  commonly 
employed. 

ESMAKCH'S  COUNTER. — Esmarch  has  devised  a 
counter  (Fig.  94)  for  estimating  the  number  of  col- 
onies present  when  they  are  upon  a  cylindrical  sur- 


BACTERIOLOGICAL  STUDY  OF  WATER.       457 

face,  as  when  in  rolled  tubes.  The  principles  and 
methods  of  estimation  are  practically  the  same  as  those 
given  for  Wolff hiigePs  apparatus.  If  the  number  of 
colonies  in  an  Esmarch  tube  is  to  be  determined,  a  sim- 
pler method  than  the  use  of  his  apparatus  may  be  em- 
ployed. It  consists  in  dividing  the  tube  by  lines  into 
four  or  six  longitudinal  areas  which  are  subdivided  by 
transverse  lines  drawn  about  1  or  2  cm.  apart.  The 
lines  may  be  drawn  with  pen  and  ink,  They  need  not 

FIG.  94. 


Esmarch's  apparatus  lor  counting  colonies  in  roll  tubes. 

be  exactly  the  same  distance  apart,  nor  exactly  straight. 
Beginning  with  one  of  these  squares  at  one  end  of  the 
tube,  which  may  be  marked  with  a  cross,  the  tube  is 
twisted  with  the  fingers,  always  in  one  direction,  and 
the  exact  number  of  colonies  in  each  square  as  it  appears 
in  rotation  is  counted,  care  being  taken  not  to  count  a 
square  more  than  once ;  the  sums  are  then  added 
together,  and  the  result  gives  the  number  of  colonies 


458  BACTERIOLOGY. 

in  the  tube.     This  method  may  be  facilitated   by  the 
use  of  a  hand-lens. 

In  all  these  methods  there  is  one  error  that  is  difficult 
to  eliminate  :  it  is  assumed  that  each  colony  represents 
the  outgrowth  from  a  single  organism.  This  is  prob- 
ably not  always  the  case,  as  there  may  exist  clumps  of 
bacteria  which  represent  hundreds  or  even  thousands  of 
individuals,  but  which  still  give  rise  to  but  a  single 
colony — this  is  usually  estimated  as  a  single  organism 
in  the  water  under  analysis. 

Where  grounds  exist  for  suspecting  the  presence  of 
these  clumps,  they  may  in  part  be  broken  up  by  shak- 
ing the  original  water  with  sterilized  sand. 

What  has  been  said  for  the  bacteriological  examina- 
tion of  water  holds  good  for  all  fluids  which  are  to  be 
subjected  to  this  form  of  analysis. 

BACTERIOLOGICAL  AIR  ANALYSIS. — Quite  a  num- 
ber of  methods  for  the  bacteriological  study  of  the  air 
exist. 

In  the  main  they  consist  either  of  allowing  air  to  pass 
over  solid  nutrient  media  (Koch,  Hesse)  and  observing 
the  colonies  which  develop  upon  the  media,  or  of  filter- 
ing the  bacteria  from  the  air  by  means  of  porous  and 
liquid  substances,  and  studying  the  organisms  thus 
obtained.  (Miguel,  Petri,  Strauss,  Wiirz,  Sedgwick- 
Tucker.) 

The  former  methods  have  given  place  almost  entirely 
to  the  latter  for  reasons  of  greater  exactness  possessed 
by  the  latter. 

In  some  of  the  methods  which  provide  for  the  filtra- 
tion of  bacteria  from  the  air  by  means  of  liquid  sub- 
stances, a  measured  volume  of  air  is  aspirated  through 
liquefied  gelatin ;  this  is  then  rolled  into  an  Esmarch 


BACTERIOLOGICAL  AIR  ANALYSIS.  459 

tube,  and  the  number  of  colonies  counted,  just  as  was 
done  in  the  water  analysis.  This  is  the  simplest  proced- 
ure. An  objection  raised  against  it  is  that  organisms 
may  be  lost,  and  not  come  into  the  calculation,  by  pass- 
ing through  the  medium  in  the  centre  of  an  air-bubble 
without  being  arrested  by  the  fluid — an  objection  that 
appears  to  have  more  of  speculative  than  of  real  value. 

FIG.  95. 


Petri's  apparatus  for  bacteriological  analysis  of  air.    The  tube 
packed  with  sand  is  seen  at  the  point  a. 

The  methods  of  filtration  through  porous  substances 
appear,  on  the  whole,  to  give  the  best  results.  Petri 
recommends  the  aspiration  of  a  measured  volume  of  air 
through  glass  tubes  into  which  sterilized  sand  is  packed. 
(Fig.  95.)  When  the  aspiration  is  finished  the  sand  is 
mixed  with  liquefied  gelatin,  plates  are  made,  and  the 
number  of  developing  colonies  counted,  the  results  giving 
the  number  of  organisms  contained  in  the  volume  of  air 
aspirated  through  the  sand. 

The  main  objection  to  this  method  is  the  possibility  of 


460  BACTERIOLOGY. 

mistaking  a  sand  granule  for  a  colony.  This  objection 
has  been  overcome  by  Sedgwick  and  Tucker,  who  employ 
granulated  sugar  instead  of  the  sand ;  this,  when  brought 
into  the  liquefied  gelatin,  dissolves,  and  no  such  error  as 
that  possible  in  the  Petri  method  can  be  made. 

SEDGWICK-TUCKER  METHOD.  —  On  the  whole,  the 
method  proposed  by  Sedgwick  and  Tucker  gives  such 
uniform  results  that  it  is  to  be  recommended  above  the 
others.  It  is  as  follows  : 

The  apparatus  employed  by  them  consists  essentially 
of  three  parts : 

(1)  A  glass  tube  of  a  special  form  to  which  the  name 
aerobioscope  has  been  given. 

(2)  A  stout  copper  cylinder  of  about  sixteen  litres 
capacity,  provided  with  a  vacuum-gauge. 

(3)  An  air-pump. 

FIG.  96. 


d  cc  Id 

The  Sedgwick-Tucker  aerobioscope. 

The  aerobioscope  (Fig.  96)  is  about  35  cm.  in  its 
entire  length;  it  is  15  cm.  long  and  4.5  cm.  in  diameter 
at  its  expanded  part ;  one  end  of  the  expanded  part  is 
narrowed  down  to  a  neck  2.5  cm.  in  diameter  and  2.5 
cm.  long.  To  the  other  end  is  fused  a  glass  tube  15 
cm.  long  and  0.5  cm.  inside  diameter,  in  which  is  to  be 
placed  the  filtering  material. 

Upon  this  narrow  tube,  5  cm.  from  the  lower  end,  a 
mark  is  made  with  a  file,  and  up  to  this  mark  a  small 
roll  of  brass- wire  gauze  (a)  is  inserted ;  this  serves  as  a 


BACTERIOLOGICAL  AIR  ANALYSIS.  461 

stop  for  the  filtering  material  which  is  to  be  placed  over 
it.  Beneath  the  gauze  (at  6),  and  also  at  the  large  end 
(c),  the  apparatus  is  plugged  with  cotton.  When  thor- 
oughly cleaned,  dried,  and  plugged,  the  apparatus  is  to 
be  sterilized  in  the  hot-air  sterilizer.  When  cool,  the 
cotton  plug  is  removed  from  the  large  end  (c),  and  thor- 
oughly dried  and  sterilized  No.  50  granulated  sugar  is 
poured  in  until  it  just  fills  the  10  cm.  (d)  of  the  narrow 
tube  above  the  wire  gauze.  This  column  of  sugar  is  the 
filtering  material  employed  to  engage  and  retain  the  bac- 
teria. After  pouring  in  the  sugar,  the  cotton-wool  plug 
is  replaced,  and  the  tube  is  again  sterilized  at  120°  C. 
for  several  hours. 

Taking  the  air  sample.  In  order  to  measure  the 
amount  of  air  used,  the  value  of  each  degree  on  the 
vacuum-gauge  is  determined  in  terms  of  air  by  means 
of  an  air-meter,  or  by  calculation  from  the  known 
capacity  of  the  cylinder.  This  fact  ascertained,  the 
negative  pressure  indicated  by  the  needle  on  exhausting 
the  cylinder  shows  the  volume  of  air  which  must  pass 
into  it  in  order  to  fill  the  vacuum.  By  means  of  the 
air-pump  one  exhausts  the  cylinder  until  the  needle 
reaches  the  mark  corresponding  to  the  amount  of  air 
required.1 

A  sterilized  aerobioscope  is  now  to  be  fixed  in  the 
upright  position  and  its  small  end  connected  by  a  rubber 
tube  with  a  stopcock  on  the  cylinder,  or  to  a  glass  tube 
tightly  fixed  in  the  neck  of  an  aspirating  bottle  by 
means  of  a  perforated  rubber  stopper.  The  cotton  plug 

1  Such  a  cylinder  and  air-pump  are  not  necessary.  A  pair  of  ordinary  as- 
pirating bottles  of  known  capacity  graduated  into  litres  and  fractions  thereof 
answer  perfectly  well.  Or  one  can  determine  by  the  weight  of  water  that  has 
flowed  from  the  aspirator,  the  volume  of  air  that  has  passed  in  to  take  its 
place,  i.  e.,  the  volume  of  air  that  has  passed  through  the  aerobioscope. 


462 


BACTERIOLOGY. 


is  then  removed  from  the  upper  end  of  the  aerobioscope, 
and  the  desired  amount  of  air  is  aspirated  through  the 
sugar.  Dust  particles  and  bacteria  will  be  held  back 
by  the  sugar.  During  manipulation  the  cotton  plug  is 
to  be  protected  from  contamination. 

FIG.  97. 


Bent  funnel  for  use  with  aerobioscope. 

When  the  required  amount  of  air  has  been  aspirated 
through  the  sugar  the  cotton  plug  is  replaced,  and  by 
gently  tapping  the  aerobioscope  while  held  in  an  almost 
horizontal  position,  the  sugar,  and  with  it  the  bacteria, 
are  brought  into  the  large  part  (e)  of  the  apparatus. 
When  all  the  sugar  is  thus  shaken  down  into  this  part 
of  the  apparatus,  about  20  c.c.  of  liquefied,  sterilized 
gelatin  is  poured  in  through  the  opening  at  the  end  c, 
the  sugar  dissolves,  and  the  whole  is  then  rolled  on  ice, 
just  as  is  done  in  the  preparation  of  an  ordinary 
Esmarch  tube. 

The  gelatin  is  most  easily  poured  into  the  aerobio- 


BACTERIOLOGICAL  STUDY  OF  THE  SOIL.     463 

scope  by  the  use  of  a  small,  sterilized,  cylindrical  funnel 
(Fig.  97),  the  stem  of  which  is  bent  to  an  angle  of  about 
110°  with  the  long  axis  of  the  body. 

The  larger  part  of  the  aerobioscope  is  divided  into 
squares  to  facilitate  the  counting  of  the  colonies. 

By  the  employment  of  this  apparatus  one  can  make 
these  analyses  at  any  place,  and  can,  without  fear  of 
contamination,  carry  the  tubes  to  the  laboratory,  where 
the  cultivation  part  of  the  work  may  be  done. 

Aside  from  this  advantage,  the  filter  being  soluble, 
only  the  insoluble  bacteria  are  left  imbedded  in  the 
gelatin. 

For  general  use  this  method  is  to  be  preferred  to  the 
others  that  have  been  mentioned. 

BACTERIOLOGICAL  STUDY  OF  THE  SOIL. — Bacterio- 
logical study  of  the  soil  may  be  made  by  either  breaking 
up  small  particles  of  earth  in  liquefied  media  and 
making  plates  directly  from  this,  or  by  what  is  per- 
haps a  better  method,  as  it  gets  rid  of  insoluble  particles 
which  may  give  rise  to  errors  :  breaking  up  the  soil  in 
sterilized  water  and  then  making  plates  immediately 
from  the  water. 

It  must  be  borne  in  mind  that  many  of  the  ground 
organisms  belong  to  the  anaerobic  group,  so  that  in 
these  studies  this  point  should  be  remembered  and  the 
methods  for  the  cultivation  of  such  organisms  practised 
in  connection  with  the  ordinary  methods.  It  must  also 
be  remembered  that  the  nitrifying  organisms,  everywhere 
present  in  the  ground,  cannot  be  isolated  by  the  ordinary 
methods,  and  will  not  appear  in  plates  made  after  either 
of  the  above  plans.  The  special  devices  that  have  been 
arranged  for  their  cultivation  will  be  found  in  the 
chapter  on  soil  organisms. 


CHAPTER  XXVIII. 

Methods  of  testing  disinfectants  and  antiseptics— Experiments  illustrating 
the  precautions  to  be  taken— Experiments  in  skin  disinfection. 

THERE  are  several  ways  of  determining  the  germicidal 
value  of  chemical  substances,  the  most  common  being 
to  expose  organisms  dried  upon  bits  of  silk  thread  to 
the  disinfectant  for  different  lengths  of  time,  and  then, 
after  removing,  and  carefully  washing  the  threads  in 
water,  to  place  them  in  nutrient  media  at  a  favorable 
temperature,  and  notice  if  any  growth  appears.  If  no 
growth  results  the  disinfection  is  presumably  successful. 
Another  method  is  to  mix  fluid  cultures  of  bacteria 
with  the  disinfectant  in  varying  proportions,  and,  after 
different  intervals  of  time,  to  determine  if  disinfection 
is  in  progress  by  transferring  a  portion  of  the  mixture 
to  nutrient  media,  just  as  in  the  other  method  of  work. 

By  the  former  process  the  bits  of  thread,  usually 
about  1  to  2  cm.  long,  are  placed  in  a  dry  test-tube 
provided  with  a  cotton  plug  and  carefully  sterilized, 
either  by  the  dry  method  or  in  the  steam  sterilizer, 
before  using.  They  are  then  immersed  in  a  pure 
bouillon  culture  or  in  a  salt  solution  suspension  of  the 
organism  upon  which  the  disinfectant  is  to  be  tested. 
I  say  pure  culture  because  it  is  always  desirable  in 
testing  a  new  germicide  to  determine  its  value  as  such 
on  several  different  resistant  species  of  bacteria,  both  in 
the  vegetating  and  in  the  spore  stage.  After  the  threads 
have  remained  in  the  culture  or  suspension  for  from  five 


METHODS  OF  TESTING  DISINFECTANTS.       465 

to  ten  minutes  they  are  removed  under  antiseptic  pre- 
cautions and  carefully  separated  and  spread  out  upon  the 
bottom  of  a  sterilized  Petri  dish.  This  is  then  placed 
either  in  the  incubator  at  a  temperature  not  exceeding 
38°  C.  until  the  excess  of  fluid  has  evaporated,  or  in  a 
desiccator  over  sulphuric  acid,  calcium  chloride,  or  any 
other  drying  agent,  but  they  are  not  left  there  until 
absolutely  dry,  only  until  the  excess  of  moisture  has 
disappeared.  When  sufficiently  dry  they  can  then  be 
employed  in  the  test.  This  is  done  by  immersing  them 
in  solutions  of  the  disinfectant  of  different  but  known 
strengths  for  a  fixed  interval  of  time,  say  one  or  two 
hours,  after  which  they  are  removed,  rinsed  off  in 
sterilized  distilled  water  to  remove  the  excess  of  disin- 
fectant adhering  to  them,  and  placed  in  fresh  steril- 
ized culture  media,  which  is  then  placed  in  the  incu- 
bator at  from  37°  to  38°  C.  If  after  twenty-four, 
forty- eight,  or  seventy-two  hours  a  growth  occurs  at  or 
about  the  bit  of  thread,  and  this  growth  consists  of  the 
organism  upon  which  the  test  was  made,  manifestly 
there  has  been  no  disinfection ;  if  no  growth  occurs 
after,  at  most,  ninety-six  hours,  it  is  safe  to  presume 
that  the  bacteria  have  been  killed,  unless  our  efforts  at 
rinsing  off  the  excess  of  disinfectant  from  the  thread 
have  not  been  successful,  and  a  small  amount  of  dis- 
infectant is  now  active  in  preventing  development,  i.  e., 
is  acting  as  an  antiseptic. 

By  the  latter  process,  in  which  cultures  or  suspen- 
sions of  the  organisms  are  mixed  with  different  but 
known  strengths  of  the  disinfectant,  a  small  portion  of 
the  mixture,  usually  a  loopful  or  a  drop,  is  transferred 
at  the  end  of  a  definite  time  to  the  fresh  medium  which 
is  to  determine  whether  the  organisms  have  been  killed 


466  BACTERIOLOGY. 

or  not.  This  is  commonly  a  tube  of  fluid  agar-agar 
which  is  poured  out  into  a  Petri  dish,  allowed  to  solidify, 
and  placed  in  the  incubator,  as  in  the  other  experiment. 

After  the  minimum  strength  of  disinfectant  necessary 
to  destroy  the  vitality  of  the  organisms  with  which  we 
are  working  has  been  determined,  for  any  fixed  time,  it 
then  remains  for  us  to  decide  what  is  the  shortest  time 
in  which  this  strength  will  have  the  same  effect.  We 
then  work  with  a  constant  dilution  of  the  disinfectant, 
but  with  different  intervals  of  exposure — one,  five,  ten 
minutes,  etc. — until  we  have  decided  not  only  the 
minimum  amount  of  disinfectant  required  for  the  de- 
struction of  the  bacteria,  but  the  shortest  time  neces- 
sary for  this  under  known  conditions. 

A  factor  not  to  be  lost  sight  of  is  the  temperature 
under  which  these  experiments  are  conducted,  for  it 
must  always  be  borne  in  mind  that  the  action  of  a  dis- 
infectant is  usually  more  energetic  at  a  higher  than  at  a 
lower  temperature. 

Now  in  both  of  these  methods  it  is  easy  to  see  that 
unless  special  precautions  are  taken  a  minute  portion  of 
the  disinfectant  may  be  carried  along  with  the  thread, 
or  drop,  into  the  medium  which  is  to  determine  whether 
the  organisms  do  or  do  not  possess  the  power  of  growth, 
and  here  have  a  restraining  or  antiseptic  action.  For 
organisms  in  their  normal  condition,  that  is,  those  which 
have  never  been  exposed  to  the  action  of  a  disinfectant, 
the  amount  necessary  to  restrain  growth,  for  certain  dis- 
infecting agents,  is  very  small  indeed,  and  for  organisms 
that  have  already  been  exposed  for  a  time  to  such  agents 
this  amount  is  even  much  less.  It  is  plain,  then,  that 
if  the  test  is  to  be  an  accurate  one,  precautions  must  be 
taken  against  admitting  this  minute  trace  of  disinfect- 


METHODS  OF  TESTING  DISINFECTANTS.      467 

ant  to  the  medium  with  which  we  are  to  determine  if 
the  bacteria  that  have  been  exposed  to  its  action  have 
been  killed  or  not. 

The  precautions  that  have  hitherto  been  taken  for 
preventing  this  accident  are,  where  the  threads  are  em- 
ployed, washing  in  sterilized  distilled  water  and  then 
in  alcohol ;  or,  where  the  fluid  cultures  were  mixed  with 
the  disinfectant  in  solution,  an  effort  was  usually  made 
to  dilute  the  amount  of  disinfectant  carried  over  to  a 
point  at  which  it  loses  its  inhibiting  power. 

While  these  are  sufficient  in  many  cases,  they  do  not 
answer  for  all.  Certain  chemicals  have  the  property 
of  combining  so  firmly  with  the  threads  upon  which  the 
bacteria  are  located  as  to  require  other  special  means 
of  ridding  the  threads  of  them ;  and  in  solutions  in 
which  proteid  substances  are  present  along  with  the 
bacteria  a  similar  union  between  them  and  the  disin- 
fectant may  likewise  take  place.  In  both  instances  this 
amount  of  disinfectant  adhering  to  the  silk  threads  or  in 
combination  with  the  proteid s  must  be  gotten  rid  of, 
otherwise  the  results  of  the  test  may  be  fallacious.  A 
partial  solution  of  the  problem  comes  from  studies  that 
have  been  made  upon  corrosive  sublimate  in  its  various 
applications  for  disinfecting  purposes,  and  in  this  con- 
nection it  has  been  shown  by  Shaefer 1  that  it  is  impos- 
sible to  rid  silk  threads  of  the  corrosive  sublimate 
adhering  to  them  by  simple  washing,  as  the  sublimate 
acts  as  a  mordant  and  forms  a  firm  union  with  the 
tissues  of  the  threads.  Braatz 2  found  the  same  to  hold 
good  for  catgut.  For  example,  he  found  that  catgut 
which  had  been  immersed  in  solutions  of  sublimate  gave 

i  Shaefer :  Berliner  klin.  Woch.,  1890,  No.  3,  p.  50. 

»  Braatz :  Centr.  f.  Bakt.  und  Parasitenkunde,  Bd.  viii.  No.  1,  p.  8. 


468  BACTERIOLOGY. 

the  characteristic  reactions  of  the  salt  after  having  been 
immersed  in  distilled  water,  which  had  been  repeatedly 
renewed,  for  five  weeks. 

He  remarks  that  a  similar  firm  combination  between 
sublimate  and  cotton  will  take  place  after  a  longer  time, 
but  it  occurs  so  slowly  that  it  cannot  interfere  with  dis- 
infection experiments  in  the  same  way  as  he  believes  the 
employment  of  silk  to  act. 

The  most  successful  attempt  at  removing  all  traces 
of  sublimate  from  the  threads  or  from  the  proteid  sub- 
stances in  which  are  located  the  bacteria  whose  vitality 
are  to  be  tested,  is  that  made  by  Geppert,  who  subjected 
them  to  the  action  of  ammonium  sulphide  in  solution. 
By  this  procedure  the  mercury  is  converted  into  in- 
soluble sulphide  and  does  not  now  have  an  inhibiting 
effect  upon  the  growth  of  those  bacteria  that  may  not 
have  succumbed  to  its  action  when  in  the  form  of  the 
bichloride. 

In  the  second  method  of  testing  disinfectants,  men- 
tioned above — that  is,  when  cultures  of  bacteria  and 
solutions  of  the  disinfectant  are  mixed,  and  after  a  time 
a  drop  of  the  mixture  is  removed  and  added  to  sterile 
nutrient  media,  the  inhibiting  amount  of  disinfectant 
can  readily  be  gotten  rid  of  by  dilution,  that  is  to  say, 
instead  of  transporting  the  drop  directly  to  the  fresh 
medium,  add  it  to  10  or  12  c.c.  of  sterilized  salt  solu- 
tion (0.6-0.7  per  cent,  of  NaCl  in  distilled  water),  or 
distilled  water,  and  after  thoroughly  shaking  add  a  drop 
of  this  to  the  medium  in  which  the  power  of  development 
of  the  bacteria  is  to  be  determined. 

Another  important  point  to  be  borne  in  mind  in 
testing  disinfectants  is  the  necessity  of  so  arranging  the 
conditions  that  each  individual  organism  will  be  ex- 


METHODS  OF  TESTING  DISINFECTANTS       469 


PIG.  98. 


posed  to  the  action  of  the  agent  used.  When  clumps 
of  bacteria  exist  we  are  not  always  assured  of  this,  for 
only  those  on  the  surface  of  the  clump 
may  be  affected,  while  those  in  the 
centre  of  the  mass  may  entirely  escape, 
being  protected  by  those  surrounding 
them.  These  clumps  and  minute  masses 
are  especially  liable  to  be  present  in 
fluid  cultures  and  in  suspensions  of 
the  bacteria,  and  must  be  eliminated 
before  the  test  is  begun,  if  it  is  to  be 
made  by  mixing  them  with  solutions 
of  the  agent  to  be  tested.  This  is 
best  accomplished  in  the  following 
way :  The  organisms  should  be  culti- 
vated in  bouillon  containing  sand 
or  finely  divided  particles  of  glass; 
after  growing  for  a  sufficient  length  of 
time  they  are  then  to  be  shaken  thor- 
oughly, in  order  that  all  clumps  may 
be  mechanically  broken  up  by  the  sand. 
The  culture  is  then  filtered  through  a 
tube  containing  closely  packed  glass 
wool. 

The  filtration  may  be  accomplished 
without  fear  of  contamination  of  the 
culture  by  the  employment  of  an 
Allihin  tube,  which  is  practically  noth-  cylindrical  funnel 

used  for  filtering  cul- 

ing  more  than  a  thick- walled  test-tube  tures  on  which  dis- 

drawn  out  to  a  finer  tube  at  its  blunt  jnff<f  nts  are  to  be 

tested. 

end  so  as  to  convert  it  into  a  sort  of 
cylindrical  funnel.     The  tube  when  finished  and  ready 
for  use  has  the  appearance  given  in  Fig.  98. 

21 


470  BACTERIOLOGY. 

The  whole  tube,  after  being  plugged  at  the  bottom 
of  its  wide  part  with  glass  wool  and  at  its  wide  open 
extremity  with  cotton  wool,  is  placed  vertically,  small 
end  down,  into  an  Erlenmeyer  flask  of  about  100  c.c. 
capacity  and  sterilized  in  a  steam  sterilizer  for  the 
proper  time.  It  is  kept  in  the  covered  sterilizer  until 
it  is  to  be  used,  which  should  be  as  soon  as  possible 
after  sterilization. 

The  watery  suspension  or  bouillon  culture  of  the 
organisms  is  now  to  be  filtered  repeatedly  through  the 
glass  wool  into  sterilized  flasks  until  a  degree  of  trans- 
parency is  reached  which  will  permit  the  reading  of 
moderately  fine  print  through  a  layer  of  the  fluid  of 
about  2  cm.  thick,  i.  e.,  through  an  ordinary  test-tube 
full  of  it.  It  can  then  be  subjected  to  the  action  of 
the  disinfectant,  and,  as  a  rule,  the  results  are  far  more 
uniform  than  when  no  attention  is  paid  to  the  exist- 
ence of  clumps.  It  is  hardly  necessary  to  say  that  in 
the  practical  employment  of  disinfectants  outside  the 
laboratory  no  such  precautions  are  taken,  but  in  lab- 
oratory work,  where  it  is  desired  to  determine  exactly 
the  value  of  different  substances  as  germicides,  all  the 
precautions  that  have  been  mentioned  will  be  found 
essential  to  success. 

In  determining  the  germicidal  value  of  different 
chemical  agents  upon  certain  pathogenic  bacteria,  sus- 
ceptible animals  are  sometimes  inoculated  with  the 
organisms  after  they  have  been  exposed  to  the  disinfect- 
ant. If  no  pathological  condition  results,  disinfection  is 
presumed  to  have  been  successful,  while  if  the  condition 
characteristic  of  the  activities  of  the  given  organism  in 
the  tissues  of  this  animal  appears,  the  reverse  is  the  case. 
The  objections  to  this  method  that  have  been  raised 


DETERMINA  TION  OF  ANTISEPTIC  PR  OPERTIES.    47 1 

are :  "Mrst.  The  test  organisms  may  be  modified  as 
regards  reproductive  activity  without  being  killed ;  and 
in  this  case  a  modified  form  of  the  disease  may  result 
from  the  inoculation,  of  so  mild  a  character  as  to  escape 
observation.  Second.  An  animal  that  has  suffered  this 
modified  form  of  the  disease  enjoys  protection,  more  or 
less  perfect,  from  future  attacks,  and  if  used  for  a  subse- 
quent experiment  may,  by  its  immunity  from  the  effects 
of  the  pathogenic  test  organism,  give  rise  to  the  mis- 
taken assumption  that  this  had  been  destroyed  by  the 
action  of  the  germicidal  agent  to  which  it  had  been  sub- 
jected." (Steruberg.) 

DETERMINATION   OF   ANTISEPTIC   PROPERTIES. 

In  this  test  sterile  media  are  employed  and  are  usually 
arranged  in  two  groups :  the  one  to  remain  normal  in 
composition  and  to  serve  as  controls,  while  to  the  other 
is  to  be  added  the  substance  to  be  tested  in  different  but 
known  strengths.  It  is  customary  to  employ  test-tubes 
each  containing  an  exact  amount  of  bouillon,  gelatin,  or 
agar-agar,  as  the  case  may  be.  To  each  tube  a  definite 
amount  of  the  antiseptic  is  added,  and  if  it  is  not  of  a  vola- 
tile nature  or  not  injured  by  heat,  they  may  then  be  ster- 
ilized. After  this  they  are  to  be  inoculated  with  the 
organism  upon  which  the  test  is  to  be  made,  and  at  the 
same  time  one  of  the  "  control "  tubes  (one  of  those  to 
which  no  antiseptic  has  been  added)  is  inoculated.  They 
are  all  then  to  be  placed  in  the  incubator  and  kept  under 
observation.  If  at  the  end  of  twenty-four,  forty-eight, 
or  seventy-two  hours  no  growth  appears  in  any  but  the 
"  control "  tubes,  it  is  evident  that  the  antiseptic  must 
be  added  in  smaller  amounts,  for  we  are  to  determine  the 


472  BACTERIOLOGY. 

point  at  which  it  is  not  as  well  as  that  at  which  it  is 
capable  of  preventing  development.  The  experiment  is 
then  repeated,  using  smaller  amounts  of  the  antiseptic 
until  we  reach  a  point  at  which  growth  just  occurs  not- 
withstanding the  presence  of  the  antiseptic,  and  its  anti- 
septic strength  falls  a  trifle  above  the  amount  present  in 
this  tube.  If,  for  example,  there  was  development  in 
the  tubes  in  which  the  antiseptic  was  present  in  the  pro- 
portion of  1:1000  and  no  growth  in  the  one  in  which  it 
was  present  in  1:1400,  the  experiment  would  be  repeated 
with  strength  of  the  antiseptic  corresponding  to  1:1000, 
1:1100,  1:1200,  1:1300,  1:1400,  and  in  this  way  one 
gradually  strikes  the  point  at  which  growth  is  just  pre- 
vented. This  point  represents  the  antiseptic  value  of 
the  substance  used,  for  the  organism  upon  which  it  has 
been  tested. 

EXPERIMENTS. 

Into  each  of  three  tubes  containing  10  c.c — one  of 
normal  salt  solution,  another  of  bouillon,  a  third  of  fluid 
blood-serum — add  as  much  of  a  culture  of  the  staphylo- 
coccus  pyogenes  aureus  as  can  be  held  upon  the  looped 
platinum  needle.  Mix  this  thoroughly,  so  that  no  clumps 
exist,  and  then  add  exactly  10  c.c.  of  1 : 500  solution  of 
corrosive  sublimate.  Mix  it  thoroughly,  and  at  the  end 
of  three  minutes  transfer  a  drop  from  each  tube  into  a 
tube  of  liquefied  agar-agar,  and  pour  this  into  a  Petri 
dish.  Label  each  dish  carefully  and  place  them  in  the 
incubator.  Are  the  results  the  same  in  all  the  plates  ? 
How  are  the  differences  to  be  explained  ?  To  what 
strength  of  the  disinfectant  were  the  organisms  ex- 
posed in  the  experiment  ? 


EXPERIMENTS.  473 

Into  each  of  two  tubes  containing  10  c.c. — the  one  of 
normal  salt  solution,  the  other  of  bouillon — add  as  much 
of  a  spore-containing  culture  of  anthrax  bacilli  as  can 
be  held  upon  the  loop  of  the  platinum  wire.  Mix  this 
thoroughly  so  that  no  clumps  exist,  and  then  add  ex- 
actly 10  c.c.  of  a  1:500  solution  of  corrosive  sublimate. 
Mix  thoroughly  and  at  the  end  of  five  minutes  transfer 
a  drop  from  each  tube  into  a  tube  of  liquefied  agar-agar. 
Pour  this  immediately  into  a  Petri  dish.  Label  each 
dish  carefully  and  place  them  in  the  incubator.  Note 
the  results  at  the  end  of  twenty-four,  forty-eight,  and 
seventy-two  hours.  How  do  you  explain  them  ? 

Make  identically  the  same  experiment  with  the  same 
spore-containing  culture  of  anthrax  bacilli,  except  that 
the  drop  from  the  mixture  is  to  be  transferred  to  10  c.c. 
of  a  mixture  of  equal  parts  of  ammonium  sulphide  and 
sterilized  distilled  water.  After  remaining  in  this  for 
about  half  a  minute,  a  drop  is  to  be  transferred  to  a  tube 
of  liquefied  agar-agar,  poured  into  Petri  dishes,  labelled, 
and  placed  in  the  incubator.  Note  the  results.  Do  they 
correspond  with  those  obtained  in  the  preceding  experi- 
ment ?  How  are  the  differences  explained  ? 

Prepare  a  1: 1000  solution  of  corrosive  sublimate.  To 
each  of  twelve  tubes  containing  exactly  10  c.c.  of  bouillon 
add  one  drop  to  the  first,  two  drops  to  the  second,  and 
so  on  until  the  last  tube  has  had  twelve  drops  added  to 
it.  Mix  thoroughly  and  then  inoculate  each  with  one 
wire-loopful  of  a  bouillon  culture  of  staphylococcus 
pyogenes  aureus.  Place  them  all  in  the  incubator  after 
carefully  labelling  them.  Note  the  order  in  which 
growth  appears. 


474  BACTERIOLOGY. 

Do  the  same  with  anthrax  spores,  with  spores  of 
bacillus  subtilis  and  with  the  typhoid  bacillus,  and  see 
how  the  results  compare.  From  these  experiments  what 
will  be  the  strength  of  corrosive  sublimate  necessary  to 
act  as  an  antiseptic  under  these  conditions  for  the  organ- 
isms employed  ? 

Make  a  similar  series  of  experiments,  using  a  5  per 
cent,  solution  of  carbolic  acid. 

Determine  the  antiseptic  point  of  the  common  disin- 
fectants for  the  organisms  with  which  you  are  working. 

Determine  the  time  necessary  for  the  destruction  of 
the  organisms  with  which  you  are  working,  by  corro- 
sive sublimate  in  1  :  1000  solution,  under  different  con- 
ditions— with  and  without  the  presence  of  albuminous 
bodies  other  than  the  bacteria,  and  under  varying  condi- 
tions of  temperature. 

In  making  these  experiments  be  careful  to  guard 
against  the  introduction  of  enough  sublimate  into  the 
agar-agar  from  which  the  Petri  plate  is  to  be  made  to 
inhibit  the  growth  of  the  organisms  which  may  not 
have  been  destroyed  by  the  sublimate.  This  may  be 
done  by  transferring  two  drops  from  the  mixture  of 
sublimate  and  organism  into  not  less  than  10  c.c.  of 
sterilized  physiological  salt  solution  in  which  they  may 
be  thoroughly  shaken  for  from  one  to  two  minutes,  or  into 
the  solution  of  ammonium  sulphide  of  the  strength  given. 

To  10  c.c.  of  a  bouillon  culture  of  staphy  loco  ecus 
pyogenes  aureus,  or  anthrax  spores,  add  10  c.c.  of  cor- 
rosive sublimate  in  1  :  500  solution,  and  allow  it  to  re- 
main in  contact  with  the  organisms  for  only  one-half  the 
time  necessary  to  destroy  them  (use  an  organism  for 


EXPERIMENTS.  475 

which  this  has  been  determined).  Then  transfer  a  drop 
of  the  mixture  to  each  of  three  liquefied  agar-agar  tubes 
and  pour  them  into  Petri  dishes.  Place  them  in  the 
incubator  and  observe  them  for  twenty-four,  forty-eight, 
and  seventy-two  hours.  No  growth  occurs.  How  is 
this  to  be  accounted  for  ? 

At  the  end  of  seventy-two  hours  inoculate  all  of 
these  plates  with  a  culture  of  the  same  organism  which 
has  not  been  exposed  to  sublimate,  by  taking  up  bits  of 
the  culture  on  the  needle  and  drawing  it  across  the 
plates.  A  growth  now  results.  We  have  here  an  ex- 
periment in  which  organisms  which  have  been  exposed 
to  sublimate  for  a  much  shorter  time  than  necessary  to 
destroy  them,  when  transferred  directly  to  a  favorable 
culture  medium  do  not  grow,  and  yet,  when  the  same 
organism  which  has  not  been  exposed  to  sublimate  is 
planted  upon  the  same  medium  it  does  grow.  How  is 
this  to  be  accounted  for  ? 

Skin-disinfection.  With  a  sterilized  knife  scrape  from 
the  skin  of  the  hands,  at  the  root  of  the  nails  and  under 
the  nails,  small  particles  of  epidermis.  Prepare  plates 
from  them.  Note  the  results. 

Wash  the  hands  carefully  for  ten  minutes  in  hot 
water  and  scrub  them  during  this  time  with  soap  and  a 
sterilized  brush.  Rinse  them  in  hot  water.  Again 
prepare  plates  from  scrapings  of  the  skin  on  the  fingers, 
at  the  root  of  the  nails,  and  under  the  nails.  Note  the 
results. 

Again,  wash  as  before  in  hot  water  with  soap  and 
brush,  rinse  in  hot  water,  then  soak  the  hands  for  five 
minutes  in  1  : 1000  corrosive  sublimate  solution,  and, 
as  before,  prepare  plates  from  scrapings  from  the  same 
localities.  Note  the  results. 


476  BACTERIOLOGY. 

Repeat  this  latter  procedure  in  exactly  the  same  way, 
but  before  taking  the  scrapings  let  some  one  pour  am- 
monium sulphide  over  the  points  from  which  the  scrap- 
ings are  to  be  made.  After  it  has  been  on  the  hands 
about  three  minutes  again  scrape,  and  note  the  results 
upon  plates  made  from  the  scrapings. 

Wash  as  before  in  hot  water  and  soap,  rinse  in  clean 
hot  water,  immerse  for  a  minute  or  two  in  alcohol,  after 
this  in  1  : 1000  sublimate  solution,  and  finally  in  am- 
monium sulphide,  and  then  prepare  plates  from  scrapings 
from  the  points  mentioned. 

In  what  way  do  the  results  of  these  experiments 
differ  one  from  another? 

To  what  are  these  differences  due? 

What  have  these  experiments  taught  ? 

In  making  the  above  experiments  it  must  be  remem- 
bered that  the  strictest  care  is  necessary  in  order  to  pre- 
vent the  access  of  germs  from  without  into  our  media. 
The  hand  upon  which  the  experiment  is  being  performed 
must  be  held  away  from  the  body  and  must  not  touch 
any  object  not  concerned  in  the  experiment.  The  scrap- 
ing should  be  done  with  the  point  of  a  knife  that  has 
been  sterilized  in  the  flame  and  allowed  to  cool.  The 
scrapings  may  be  transferred  directly  from  the  knife- 
point to  the  gelatin  by  means  of  a  sterilized  platinum 
wire  loop. 

The  brush  used  should  be  thoroughly  cleansed  and 
always  kept  in  1  : 1000  solution  of  corrosive  sublimate. 
It  should  be  washed  in  hot  water  before  using. 


APPENDIX. 


LIST  of  apparatus  and  materials  required  in  a  be- 
ginuers's  bacteriological  laboratory  : 

MICROSCOPE   AND   ACCESSORIES. 

Microscope  with  coarse  and  fine  adjustment  and 
heavy,  firm  base ;  Abbe  sub-stage  condensing  system, 
arranged  either  as  the  "  simple  "  or  as  the  regular  Abbe 
condenser,  in  either  case  to  be  provided  with  iris  dia- 
phragm ;  objectives  equivalent,  in  the  English  nomen- 
clature, to  about  one-fourth  inch,  and  one-sixth  inch 
dry,  and  one-twelfth  inch  oil-immersion  system  ;  a  triple 
revolving  nose-piece  ;  three  oculars,  varying  in  mag- 
nifying power  ;  and  a  bottle  of  immersion  oil. 

Glass  slides,  English  shape  and  size  and  of  colorless 
glass. 

Six  slides  with  depressions  in  centre  of  about  6  to  8 
mm.  in  diameter. 

Cover-slips,  15  by  15  mm.  square  and  from  0.15  to 
0.18  mm.  thick. 

Forceps.  One  pair  of  fine-pointed  forceps  and  one 
pair  of  the  Cornet  or  Stewart  pattern,  for  holding  cover- 
slips. 

Platinum  needles  in  glass  handles.  One  straight,  of 
about  4  cm.  long ;  one  looped  at  the  end  and  of  about 
4  cm.  long  ;  and  one  straight  of  about  8  cm.  long. 

21* 


478  BACTERIOLOGY. 

Glass  handles  to  be  of  about  3  mm.  thickness  and  from 
15  to  17  cm.  long. 

STAINING  AND  MOUNTING  REAGENTS. 

200  c.c.  of  saturated  alcoholic  solution  of  fuchsiu. 

200  c.c.  of  saturated  alcoholic  solution  of  gentian 
violet. 

200  c.c.  of  saturated  alcoholic  solution  of  methylene- 
blue. 

200  grammes  of  pure  aniline. 

200  grammes  of  C.  P.  carbolic  acid. 

500  grammes  of  C.  P.  nitric  acid. 

500  grammes  of  C.  P.  sulphuric  acid. 

200  grammes  of  C.  P.  glacial  acetic  acid. 

1  litre  of  ordinary  93-95  per  cent,  alcohol. 

1  litre  of  absolute  alcohol. 
500  grammes  of  ether. 

500  grammes  of  pure  xylol. 
50  grammes  of  Canada  balsam  dissolved  in  xylol. 
100  grammes  of  Schering's  celloidin. 
10  grammes  of  iodine  and  30  grammes  of  iodide  of 
potassium  in  substance. 
100  grammes  of  tannic  acid. 
100  grammes  of  ferrous  sulphate. 
Distilled  water. 

FOR   NUTRIENT   MEDIA. 

J-  pound  Liebig's  or  Armour's  beef  extract. 
250  grammes  Witte's  peptone. 

2  kilogrammes  of  gold  label  gelatin  (Hesteberg's). 
100  grammes  of  agar-agar  in  substance. 


GLASSWARE.  479 

200  grammes  of  sodium  chloride  (ordinary  table  salt). 

500  grammes  of  pure  glycerin. 

50  grammes  of  pure  glucose. 

20  grammes  of  pure  lactose. 

100  grammes  of  caustic  potash. 

200  c.c.  of  litmus  tincture. 

10  grammes  of  rosolic  acid  (corallin). 

Blue  and  red  litmus  paper ;  curcuma  paper. 

5  grammes  of  phenolphthalein  in  substance. 

Filter  paper,  the  quality  ordinarily  used  by  druggists. 
100  grammes  of  pyrogallic  acid. 
1  kilogramme  C.  P.  granulated  zinc. 

GLASSWARE. 

200  best  quality  test-tubes,  slightly  heavier  than 
those  sold  for  chemical  work,  of  about  12  to  13  cm. 
long  and  12  to  14  mm.  inside  diameter. 

15  Petri  double  dishes  of  about  8  or  9  cm.  in  diam- 
eter and  from  1  to  1.5  cm.  deep. 

6  Florence  flasks,  Bohemian  glass,  1000  c.c.  capacity. 
6  Florence  flasks,  Bohemian  glass,  500  c.c.  capacity. 
12   Erlenmeyer  flasks,    Bohemian     glass,    100   c.c. 

capacity. 

1  graduated  measuring  cylinder,  1000  c.c.  capacity. 

1  graduated  measuring  cylinder,  100  c.c.  capacity. 

25  bottles,  125  c.c.  capacity,  narrow  necks,  with 
ground  glass  stoppers. 

25  bottles,  125  c.c.  capacity,  wide  mouths,  with  ground 
glass  stoppers. 

1  anatomical  or  preserving  jar,  with  tightly  fitting 
cover,  of  about  4  litres  capacity,  for  collecting  blood- 
serum, 


480  BACTERIOLOGY. 

2  battery  jars  of  about  2  litres  capacity,  provided 
with  loosely  fitting,  weighted,  wire-net  covers,  for  mice. 

10  feet  of  soft  glass  tubing,  2  or  3  mm.  inside  diameter. 

20  feet  of  soft  glass  tubing,  4  mm.  inside  diameter. 

6  glass  rods,  18  to  20  cm.  long  and  3  or  4  mm.  in 
diameter. 

6  pipettes  of  1  c.c.  each,  divided  into  tenths. 

2  pipettes  of  10  c.c.  each,  divided  into  cubic  centi- 
metres and  fractions. 

1  burette  of  50  c.c.  capacity,  divided  into  cubic  centi- 
metres and  fractions. 

1  separating  funnel  of  750  c.c.  capacity,  for  filling 
tubes. 

2  glass  funnels,  best  quality,  about  15  cm.  in  diam- 
eter. 

2  glass  funnels,  best  quality,  about  8  cm.  in  diameter. 
2  glass  funnels,  best  quality,  about  4  or  5  cm.    in 
diameter. 

6  ordinary  water  tumblers  for  holding  test-tubes. 
1  ruled  plate  for  counting  colonies. 

1  gas  generator,  600  c.c.  capacity,  pattern  of  Kipp  or 
v.  Wartha. 

BURNERS,    TUBING,    ETC. 

2  Bunsen  burners,  single  flame. 
1  Rose  burner. 

1  Koch  safety  burner,  single  flame. 
6  feet  of  white  rubber  gas-tubing. 
12  feet  of  pure,  red  rubber  tubing  of  5  to  6  mm.  inside 
diameter. 

1  thermo-regulator,  pattern  of  L.  Meyer  or  Reichert. 

2  thermometers,    graduated    in   degrees    Centigrade, 
registering  from  0°  to  100°,  graduated  on  the  stem. 


INCUBATORS  AND  STERILIZERS.  481 

1  thermometer  graduated  in  tenths  and  registering 
from  0°  to  50°  C. 

1  thermometer  registering  to  200°  C. 

INSTRUMENTS,    ETC. 

1  microtome,  pattern  of  Schanze,  with  knife. 

1  razor  strop. 

6  cheap  quality  scalpels,  assorted  sizes. 

2  pair  heavy  dissecting  forceps. 

1  pair  medium-size  straight  scissors. 
1  pair  small-size  straight  scissors. 

1  hypodermic  syringe  that  will  stand  steam  steriliza- 
tion. 

2  teasing  needles. 

1  pair  long-handled  crucible  tongs  for  holding  mice. 

1  wire  mouse-holder. 

2  small  pine  boards  on  which   to   tack  animals   for 
autopsy. 

2  covered  stone  jars  for  disinfectants  and  for  receiving 
infected  materials. 

INCUBATORS   AND   STERILIZERS. 

1  incubator,  simple  square  form,  either  entirely  of 
copper,  or  of  galvanized  iron  with  copper  bottom. 

1  medium-size  hot-air  sterilizer  with  double  walls, 
asbestos  jacket,  and  movable  false  bottom  of  copper 
plates. 

1  medium-size  steam  sterilizer;  either  the  pattern 
of  Koch,  or  that  known  as  the  Arnold  steam  sterilizer, 
preferably  the  latter. 


482  BACTERIOLOGY. 

MISCELLANEOUS. 

1  pair  of  balances,  capacity  1  kilogramme ;  accurate 
to  0.2  gramme. 

1  set  of  cork  borers. 
1  hand-lens. 

1  wooden  filter-stand. 

2  iron  stands  with  rings  and  clamps. 

3  round,  galvanized  iron  wire  baskets  to  fit  loosely 
into  steam  sterilizer. 

3  square,  galvanized  iron  wire  baskets  to  fit  loosely 
into  hot-air  sterilizer. 

1  sheet-iron  box  for  sterilizing  pipettes,  etc. 

1  covered,  agate-ware  saucepan,  1200  c.c.  capacity. 

2  iron  tripods. 

1  yard  of  moderately  heavy  wire  gauze. 

2  test-tube  racks,  each  holding  24'tubes,  12  in  a  row. 

1  constant-level,  cast-iron  water-bath. 

2  potato  knives. 

2  test-tube  brushes  with  reed  handles. 

Cotton  batting. 

Copper  wire,  wire  nippers. 

Round  and  triangular  files. 

Labels. 

Towels  and  sponges. 


INDEX. 


Abbe,  substage   condensing  sys- 
tem of,  26 
Abscess,  production  of,  238,  239 

histological  study  of,  239-241 
Aerobic  bacteria,  33 
Aerobioscope,  460 
Agar-agar,  preparation   of     (see 

Media). 

properties  of,  74-76 
Air,  bacteriological   analysis   of, 

458-463 

Petri's  method  for,  459, 
.      460 

Sedgwick-Tucker  meth- 
od, 460-463 
Alexines,  420,428 
Anaerobic  bacteria, 

methods  of  cultivating,  185- 

191 

Buchner's,  187 
Esmarch's,  190, 191 
Frankel's,  187, 188 
Hesse's,  186 

Kitasato  and  Weil's,  190 
Koch's,  186 
Liborius's,  186 
Aniline  dyes  for  differentiating 

bacteria,  181 

Animals,   fluctuations  in  weight 
and  temperature  of,  212- 

218 
inoculation  of,  197-218 

apparatus  used  in,  199- 

202,  205,  207 
intralymphatic,  208 
intraocular,  211 
intraperitoneal  and  pleu- 

ral,  209-211 
intravascular,   203-208 
subcutaneous,  197-203 
observations  of,  after  inocula- 
tion, 211-218 


Animals,  post-mortem  examina- 
tion of,  219-224 
cultures  from  tissues  at, 

221,  222 

disinfection    of    imple- 
ments after,  223,  224 
disposal  of  remains  from, 

223 

external  inspection,  219 
incision    through    skin, 

219,  220 
Nu  Wall's  spear  for  use 

at,  221,  222 

opening  the  body  cavi- 
ties, 220,  221 
position  of  animal,  219 
precautions  during,  219, 

220 
preservation  of    tissues 

from,  222,  223 
Anthrax,  376-388 

animals  that  are  susceptible 

to,  384 
bacillus  of,  376-388 

biology  of,  377-381 
discovery  of,  17,  376 
experiments  with,  384- 

388 
morphology    of,    376- 

379 
pathogenesis   of,    382- 

384 
spore  formation  of,  377, 

379 

staining  of,  381,  382 
Antiseptic,  definition  of,  70 
Antiseptics,  tests  of,  471,  472 
Apparatus   necessary  to  bacteri- 
ological work,  477-482 
preparation  of,  104-107 
Appendix,  list  of  apparatus,  477- 
482 


484 


INDEX. 


"DACILLI,  36-38 

D    differentiation  from  spores, 40 

flagellaupon,  45 

involution  forms  of,  39 

life  cycle  of,  38 

mode  of  multiplication,  41- 
44 

motility  of,  45 

spore  formation  in,  38,  39 
Bacillus  anthracis,  376,  377 

coli  communis,  322-329 

"comma,"  330-358 

diphtheria,  296-311 

Finkler-Prior,   359-364 

leprse,  282-284 

mallei  (of  glanders),  287-295 

Neapolitans,  322-329 

nitrifying,  391-394 

cedematis  maligni,  402-407 

pyocyanus,  248-253 

pseudo-diphtheria,  310 

smegma,  282-284 

subtilis,  231 

symptomatic  anthrax,   407- 
413 

syphilis,  282-284 

tetani,  395-402 

tuberculosis,  276-284 

typhi  abdominalis,  312-321 
Bacteria,  aerobic,  33 

anaerobic,  83 

methods  of  cultivating, 
185-191 

behavior  toward  staining  re- 
agents, 182 

capsule  surrounding,  145 

chromogenic,  30 

classification  of,  36 

conditions       necessary       to 
growth  of,  35 

constancy  in  morphology  of, 
39 

definition  of,  27 

denitrifying,  30 

discovery  of,  13-15 

facultative,  34 

fermentation  by,  182-185 
apparatus    for     testing, 

184 
gases  resulting  from,  185 

flagellated  forms  of,  45 

identification  of,  170 


Bacteria,  involution  forms  of,  39 
isolation  of,  in  pure  culture, 

71-76 

principles  of,  72-74 
on  slanted  media,  118, 

119 
microscopic  examination  of, 

171-179 
modes  of  multiplication   of, 

41-43 

morphology  of,  36-46 
motility  of,  45,  46 
nitrifying,  30,  391-394 
nutrition  of,  31-33 
photogenic,  30 
points  to  be  observed  in  de- 
scribing, 194-196 
reaction  produced  by,  181 
relation  to  man,  28,  29 
relation  to  temperature,  34, 

oo 

results  of  growth,  29,  30 
role  in  nature,  28 
saprogenic,  30 
spore    formation   of,  38-41, 

43,  44 

staining,  reactions  of,  182 
systematic  study  of,  170 
thermophilic,  34,  35 
thiogenic,  30 
zymogenic,  30 

Bacteriology,  application  of  meth- 
ods of,  225 
Bacterium  coli  commune,   322- 

329 

characteristics      of, 
cultural,    324- 

326 
morphological, 

323 
pathogenic, 

327-329 

differentiation     of, 
from    bac.    typh. 
abdom.,  326 
where  found,  322 
Behring  and  Kitasato,  430 
Billroth,  23 

and  Tiegel,  24 
Birch-Hirschfeld,  22 
Black  leg  (see  Symptomatic  An- 
thrax). 


INDEX. 


485 


Blood-serum  as  culture  medium 

(see  Media), 
germicidal   element  of, 

427,  428 

action  of,  425-427 
Bolton's  potato  method,  89 
Bonnet,  20 
Booker's    modification     of    Es- 

march's  method,  116 
Bouillon  (see  Media). 
Brieger  and  Cohn,  420 
Brooding  oven,  120,  121 
Brownian  motion,  176 
Buchner,  427,  431 
Bulb  for  water  samples,  449 
Burdon-Sanderson,  25 
Burner,   Koch's   safety,   for   use 
with  incubator,  122,  123 


acid   as  disinfect- 
J    ant,  68 
Chauveau,  421 
Chevreul  and  Pasteur,  19 
Chlorophyll,  27,  28 
Cholera    Asiatica,   diagnosis    of, 

OXO     QKU 

oOZ— oOo 

method  of  Schotte- 

lius,  339,  340 
spirillum  of,  330,  358 

behavior  of  in  but- 
ter, 350 

in    milk,    349,  350 

in  soil,  348 

in  water,   346,  347 

characteristics      of, 
cultural, 
333-341 
morphological, 
331-333 

destiny  of,  in  dead 
body,  349-351 

effects  of  drying, 
351 

existence  outside  the 
body,  346 

experiments     upon 
animals    with, 
341-345 

general  considera- 
tions upon,  345- 
352 


Cholera,    Asiatic,    spirillum    of, 
location    in    the 
body,  345,  346 
poisons      produced 

by,  340,  341 
relation    to    gases, 
352 

to  other  bac- 
teria, 350, 
351 

to    putrefac- 
tion, 347,  349 
to  sunlight,  348 
specific  reaction  of 
immunified    ani- 
mals, 345 

|  Chromogenic  bacteria,  30 
Classen,  23 
Cohn,  21 
Colon    bacillus   (see    Bact.    Coli 

Commune). 
Colonies,  counting  of,   454-458, 

study  of,  128-130 
Comma     bacillus    (see  Cholera, 

Asiatica). 
Cornet,  274 

Corrosive  sublimate  as  disinfect- 
ant, 65,  66 
Cooling  stage,  111 
Cover-slips,  cleaning  of,  134 
impression,  138 
microscopic      examina- 
tion of,  173,  174 
preparation  of,  134-138 
steps   in   making,    135- 

138 

Cultures,  gelatin,  179 
hanging-drop,  175 
potato,  180 
pure,  130 
reactions  of,  181 
stab  and  smear,  130, 132 
Cygnseus,  317 


HECOLOKIZ  ING  solutions,  154 
L'     Decomposition,  27,  389,  390 
Defensive  proteids,  420 
Deneke's  cheese    spirillum   (see 

Spirillum  tyrogenum). 
Denitrifying  bacteria,  30 
i  Diphtheria,  bacillus  of,  296-311 


486 


INDEX. 


Diphtheria,  bacillus,  cultural,  pe- 
culiarities of,  300-304 
experiments  upon,  310, 

311 

location  in  tissues,  306 
method  of  obtaining,  296 
modification     in     viru- 
lence, 308-311 
morphology  of,  298-300 
pathogenesis    of,    304- 

311 
poison  produced  by,  308 

potency  of,  420 
principles  of  immunify- 

ing  against,  430,  431 
pseudo-diphtheria  bac., 

308-311 

histo logical  changes  accom- 
panying, 307 
Diplococci,  38 

Disinfectants  and  antiseptics,  ex- 
periments with,  472-475 
general  considerations,  63-70 
methods  of  testing,  464-471 
precautions   to    be   ob- 
served, 466-470 
use  of  animals  as  test-objects 

for,  470,  471 

use  in  the  laboratory,  68-70 
Disinfection,   general   considera- 
tions, 63-70 
influence  of  temperature  on, 

66 

inorganic  salts  in,  66-68 
in  the  laboratory,  68-70 
modus  operandi,  65 
reliable  agents  for  purposes 

of,  68-70 
selection    of    agents    to    be 

used  in,  64,  65 
Dunham's  sol.,  100 


E BERTH,  23 
Ehrlich,  23 
Emmerich  and  Fowitzky,  436 

and  Mattei,  432 
Erysipelas,  246 
Escherich,  322 
Esmarch's  tubes,  115-118 

Booker's  method  of  roll- 
ing, 116 


Esmarch's  tubes,  made  of  agar- 
agar,  118 

Exposure  and  contact,  experi- 
ments upon,  227,  228 


FACULTATIVE,  bacteria,  34 
r     use  of  the  term,  34 
Fehleisen,  23 
Fermentation,  27,  182-185 

gases   resulting   from,   184- 

185 

particular  forms  of,  30 
tube,  184 

method  of  using,  183-185 
Filter,  method  of  folding,  82,  83 
Finkler-Prior  bacillus,  359-364 
Flagella,  45,  46 

methods  of  staining,  148-153 
Bunge's,  151 
Lceffler's,  148-151 
van   Ermengem's,   151- 

153 

Flagellated  organisms,  45 
Frankland,  G.  and  P.  F.,  391 
Funnel  for  filling  aerobioscope, 

462 

for  filling  test-tubes,  106 
for  filtering  cultures,  469 
hot-water,  83,  86 


P  AS-PRESSURE       regulator, 
U     126 

Gelatin,  cultures  in,  179, 180 

their  characteristics,  179 

180 

preparation  of  (see  Media), 
properties,  74-76 
Geppert,  65,  66,  468 
Glanders,  287-295 

bacillus  of,  289-292 

cultivation  of,  290,  291 
inoculation  with,  292 
morphology,  289,  290 
staining  of,   in   tissues, 

293,  294 
diagnosis  of,  by  use  of  mal- 

lein,  295 

by  Strauss's  method,  295 
manifestations  of,  287,  288 
histology  of,  288 


INDEX. 


487 


Glanders,  susceptibility   of  ani- 
mals to,  291 
synonyms,  287 
Gonococcus,  235 
Gonorrhoea,  pus  of,  235 
Green- pus,  bacillus,  248-253 
Guarniari's  agar-gelatin,  103 


HALSTED,  209,  238 
Hanging-drop,  175-177 
Hankin,  429 

and  Martin,  428 
Henle,  17,  18 
Hoffmann,  19 
Hot-water  funnel,  83,  86 
Hydrogen,  test  for  purity  of,  189 
Hypodermic  syringes    and   nee- 
dles, 204-208 


Infection,  conclusions  concerning, 

420 

modus  operandi,  420 
poisons  present  in,  418-420 
study  of  types  of,  414-418 
Inoculation  of  animals,  197-218 
intraocular,  211 
intraperitoneal  and  pleural, 

209-211 

intravascular,  203-208 
subcutaneous,  197-203 
intralymphatic,  208 
apparatus  used  in,  199-202, 

205,  207 

Introduction,  14-26 
Involution  forms  of  bacteria,  39 
\  Isolation  of  colonies  on   slanted 
media  in  tubes,  118,  119 


IMBEDDING  of   tissues,   158, 
1     159 

Immunity,  421-438 
acquired,  421 
conclusions  concerning,  436- 

438 

earlier  studies  on  blood,  rela- 
tive to,  425-427 
"  exhaustion  "     hypothesis, 

423 
experiments   of    the   Klem-  i 

perers  on,  432-435 
humoral  theory  of,  425 
hypothesis  of  Buchner,  431,  ! 

436 
evidence    in    favor    of, 

431-435 
natural,  421 
nature  of  protective  bodies,  i 

428,  429 
observations  of  Behring  and 

Kitasato,  430,  431 
"  retention  "  hypothesis,  421 
theory  of  Metchnikoff,  424 
Incubator,  120-122 

burner  for  heating,  122,  123 
Indol,   production    by    bacteria, 

191, 192 

method  of  detecting,  192-19 1 
Infection,  414-420 

chemical  nature  of,  418-420 


OKDAN  and  Kichards,  391 


KLEBS,  23-25 
Klemperer,  F.  and  G.,  work 

on  pneumonia,  432-435 
Koch,  fundamental  researches  of, 

25,  26 

postulates  of,  276 
safety  burner  of,   122-123; 
277,  330,  342,  343, 346,347, 
353,  354,  355,  etc. 


LACTOSE-LITMUS    agar-agar 
or  gelatin  (see  Media). 
Leeuwenhoek,  13-16 
Lens  for  counting  colonies,  456 
Lepra  bacillus,  282-284 

staining  peculiarities  of,  283, 

284 

Letzerich,  23 
Levelling  tripod,  110 
Lime,  chloride  of,  70 

milk  of,  68,  69 
Litmus  milk,  99 
Loeffler's      alkaline     methylene- 

blue,  141 

blood-serum    mixture,    102, 
103 


488 


INDEX. 


Lceffler's  discovery  of  the  bacillus 
of  diphtheria,  298 

stain  for  flagella,  46, 148, 149 
Lceffler  and  Schiitz,  discovery  of 

the  bacillus  of  glanders,  289 
Lukomsky,  23 


MALIGNANT  oedema,  bacillus 
of,  402-407 

cultural  peculiarities  of, 

404,  405 

morphology  of,  403 
pathogenesis  of,  405-407 
susceptibility  of  animals 

to,  405 
Mallein,  295 
Meat  extracts  in  culture  media,  80 

infusion,  103 
Media,  culture,  77-103 

agar-agar,  85-88 

clarification  of, 

88 
filtration  of,  85, 

86 

glycerin,  87 
neutralization 

of,  85 
solution  of,  85- 

87 

blood-serum,  91-98 
Councilman  -  Mai- 
lory  method,  95, 
96 
mixture  of  Lreffler, 

102,  103 
Nuttall's     method, 

96-98 
original  method  of 

Koch,  91-95 
preservation  of,  95 
by  chloroform, 

98 

sterilization  and  so- 
lidification of,  93- 
95 
bouillon,  77-80 

neutralization      of, 

78,79 
gelatin,  80-85 

clarification  of,  83 
filtration  of,  83-84 


Media  culture,  gelatin,  solution 

of,  81 -84 
sterilization  of,  84- 

85 
Guarniari's       agar-agar 

gelatin,  103 
lactose-litmus  agar-agar 

or  gelatin,  101,  102 
litmus  milk,  99 
meat  infusion,  103 
milk,  98-100 

-agar-agar,  99 
peptone  solution,  Dun- 
ham's, 100 

rosolic  acid,  peptone  so- 
lution, 101 
potatoes,  88 

Bolton's  method,  89, 

90 
Esmarch's  method, 

90 

mashed,  90 
original  method,  88, 

89 

Metchnikoff,  424 
Milk  (see  Media). 
Micrococci,  36,  37 

mode  of  multiplication,  41 , 42 
Micrococcus      lanceolatus,     256, 

257-262 

irregularities  in  devel- 
opment, 261 
morphological  peculiar- 
ties,  259 
results    of    inoculation 

with,  261,  262 
staining  of,  261 
susceptibility  of  animals 

to,  262 
variations  in  virulence, 

261 

where  found,  259 
Micrococcus  tetragenus,  256,  262- 

265 
cultural  peculiarities  of 

263,  264 

morphology  of.  263 
susceptibility  of  animals 

to,  265 

where  found,  263 
Microscope,  parts  of,  171-173 
Microtome,  157 


INDEX. 


489 


\TAGELI,  31 

11     NassilofF,  23 
Needham,  18 
Nitrification,  390 
Nitrifying  bacteria,  390,  391,  394 
Nitrites,  test  for,  194 
Nitro-monas     of     Winogradsky, 
391-394 

cultural  peculiarities  of, 
392-394 

morphology  of,  391,  392 
Normal  solution,  187 
Nuttall,  221,  421,  425 


OIL  immersion  system,  use  of, 
174 

construction  of,  172-173 
Oertel,  23,  307 
Ozanam,  17 


PARASITE,  27 

1      Pasteur,  17,  19,  25,  421,  423 
Peptone,  test  of  purity  of,  100 
with  rosolic  acid,  101 
Peritonitis,   production    of,    237, 

238 

Petri's  dishes,  114,  115 
Pfeiffer,  344,  358,  372 
Phagocytosis,  424 
Photogenic  bacteria,  30 
Plates,   apparatus    employed    in 

making,  109-113 
Esmarch's  modification,  115, 

116 
Booker's    modification 

of,  116,  117 

Koch's  fundamental    obser- 
vations, 71,  72 
materials  used    in   making, 

108 

Petri's  modification,  114, 115 
principles  involved,  71-76 
technique   of  making,  108- 

115 

Platinum  needles  and  loops,  109 
Plenciz,  16 

Post-mortem  examination  of  ani- 
mals, 219-224 
cultures  from  tissues  at, 
221,222 


Post-mortem,  disinfection  of  im- 
plements   after,    223, 
224 
disposal  of  remains  from, 

223 
external    inspection  at, 

219 
incision     through     the 

skin  at,  219-220 
Nuttall's  spear  for  use 

at,  221,  222 

opening  of  the  body  cav- 
ities, 220,  221 
position  of  animal  dur- 
ing, 219 
precautions  during,  219, 

220 

preparation    of    cover- 
slips  at,  222 

preservation    of    mate- 
rials, 222,  223 
Postulates  of  Koch,  276 
Potato,  characteristics  of  cultures 

on,  180 

preparation  for  culture  pur- 
poses (see  Media). 
Prudden,  271 

Pseudo-diphtheria  bacillus,  SOS- 
SI  1 

tuberculosis,  285,  286 
Pure  culture,  130 
Pus,  microscopic  appearance  of, 

234,  235 
Putrefaction,  27 
Pyaemia  production  of,  238 
Pyocyanus,  bacillus,  248-253 
chameleon  phenomenon   of, 

251 
pathogenic  properties  of,251, 

252 
protective  properties  of,  252 


Q~  UARTER  evil  or  quarter  ill 
(see  Symptomatic  Anthrax). 


DECKLINGHAUSEN,  22, 23 

It  Regulator,  gas-pressure,  126, 

127 

thermo,  124 
Rindfleisch,  22 


490 


INDEX. 


Kosolic  -  acid  -  peptone  solution 

(see  Media). 
Roux  and  Yersin,  420 


O  APROPHYTE,  27 
O     role  in  nature,  28 
Saprogenic  bacteria,  30 
Sarcinae,  38 

mode  of  multiplication,  42 
Schottelius's  method  of  examin- 
ing cholera  evacuations,  339, 
340 

Schroeder  and  Dusch,  19 
Schulze,  19 
Schwann,  19 
Section  cutting,  156,  157 
Septicaemia,  256-265 

from  micrococcus  tetragenus, 

262-265 

from  sputum,  257-262 
Skin   disinfection,    experiments, 

475,  476 

Smear  cultures,  131 
Smegma  bacillus,  staining  pecu- 
liarities, 282,  283 
Soil,  bacteriological   analysis   of, 

463 

nitrifying  bacteria  in,  390 
organisms  present  in,  394 
phenomena  in  operation  in, 

389-391 

Spallanzani,  18,  19 
Spirilla,  36-41 
Spirillum  of  Asiatic  cholera  (see 

Cholera). 

of  Deneke,  364-368 

biology  of,  364-367 

morphology  of,  364 

pathogenesis  of,  367 

of  Finkler-Prior  (see  Vibrio 

Proteus 1. 
of    Metchnikoff  (see  Vibrio 

Metchnikovi). 
of  Miller,  368-371 

biology  of,  368-371 

morphology  of,  368 

pathogenesis  of,  371 

tyrogenum  (see  Spirillum  of 

Deneke). 
undula,  45 
Spores,  formation  of,  117 


Spores,  formation  of,  method  of 

studying,  177-179 
mode  of  development,  43,  44 
recognition  of,  40 
staining  of,  146-148 
Sputum,   inoculations  with,  256, 

257 
microscopic  examination  of, 

256 

pathogenic  properties  of,  261 
septicaemias,  256 
tuberculosis,  255 
tubercular,  254,  255 
Stab  cultures,  130 
Staining,  methods  and  solutions 

used  in,  133-170 
acetic  acid,  146 
Bunge's,  151 
Gabbett's,  144 

general  remarks  on,  153, 154 
Gram's,  145 
Gray's,  162 
Koch-Ehrlich's,  141 
Kuehne's,  164 
Lceffler's  blue,  141 
Lceffler's  flagellar,  148-151 
Mo3ller's,147,  148 
ordinary  solutions  used,  139- 

141 

bottles  for  holding,  140 
van  Ermengem's,  151-153 
Weigert's,  165 
Ziehl-Neelsen,  141 
Staphylococcus    pyogenes  afbus 

242 
aureus,  237-242 

cultural    peculiari- 
ties of,  236,  237 
pathogenesis,  237 
where    to     be    ex- 
pected, 238 
citreus,  242 

Sterilization,  chemical,  63-70 
direct,  56-58 

experiments  upon,  229-233 
by  heat,  49-62 

principles  involved,  50, 

51 
by  hot  air,  62,  63 

apparatus  used,  62 
by  steam,  58-61 

apparatus  used,  58-60 


INDEX. 


491 


Sterilization  by  steam,  under  pres- 
sure, 60,  61 
intermittent,  52-54 

at  low  temperature,  55 
principles  involved,  47-70 
use  of  the  term,  47-49 
Sternberg,  259,374,471 
Strauss's  method  for  diagnosis  of 

glanders,  295 
Streptococci,  38 

mode  of  multiplication,  42 
Streptococcus  pyogenes,  242-247 
biology  of,  243-246 
effects    of     inoculation 

with,  246 

morphology  of,  242,  243 
where   to    be  expected, 
242-246 

Subtilis,  bac.,231 
Suppuration,  234-248 

bacteria  common  to.  234 
experiments  on,  237-239 
general  remarks  upon,  247- 

248 
less  common  causes  of,  242, 

247,  248 
microscopic    appearance   of 

pus,  234-235 

Symptomatic  anthrax,  407-413 
bacillus  of,  407-413 

biology  of,  409-412 
differentiation  from  ba- 
cillus    of    malignant 
osdema,  413 

morphology  of,  408,  409 
pathogenesis,  412,  413 
susceptibility  of   animals  to, 

413 

Syphilis    bacillus,    staining    of, 
282-284 


TEST-TUBES,  cleaner  for,  104 

1      cleaning  of,  104 

filling  with  media,  105,  106 

apparatus  for,  106 
plugging  with  cotton,  105 
position  after  filling,  106,  107 
sterilization  of,  105 

Tetanus,  bacillus  of,  395-402 
biology  of,  397-400 
effects  on  animals,  400,  401 


Tetanus,    method    of   obtaining, 

395-397 

morphology  of,  397 
poison  produced  by,  401,  402 
Tetanus  toxin,  potency  of,  420 
Tetrads,  38 

Thermophilic  bacteria,  30,  54, 55 
Thermo-regulator,  1 23-126 
Thermostat,  120 
Thiogenic  bacteria,  30 
Tissues,  cultures  from,  at  autop- 
sies, 221-223 
Nuttall's  spear  for  mak- 
ing, 222 

cutting  sections  of,  156-158 
hardening  of,  156 
imbedding  of,  158,  159 
in  celloidin,  158 
in  paraffin,  158,  159 
preservation  of,  156 
staining  of  bacteria  in,  159- 

169 

special  methods,  162-169 
dahlia,  163 
dry,  166 
Ehrlich's,  166 
Gram's,  162 
Gray's,  166-169 
Kuehne's,  164 
Weigert's,  165 
Ziehl-Neelsen's,166 
steps  in  the  process,  161, 

162 

Toxaemia,  417-418 
Traube  and  Gscheidlen,  425 
Treviranus,  18 

Tripod  for  levelling  plates,  110 
I  Tuberculin,  284 
!  Tuberculosis,  266-286 

cavity  formation  in,  270,  271 
conditions   simulating,    285, 

286 

diffuse  caseation  of,  269,  270 
encapsulation   of  tubercular 

foci,  271,  272 
giant  cells  in,  269 
location   of  bacilli  in,   275, 

276 

manifestations  in  experimen- 
tal, 267 

miliary  tubercles    structure 
of,  268,  269 


492 


INDEX. 


Tuberculosis,  modes  of  infection, 

273-275 

primary  infection,  272,  273 
pseudo,  '285,  286 
sputum  in.  254,  255 

inoculation  of    animals 

with,  254-257 
microscopic  appearance 

of,  256 

staining  of,  254 
susceptibility  of  animals  to, 

285 

Tuberculosis,  bacillus  of,  276-284 
appearance  of  cultures, 

279 

cultivation  from  tissues, 
277-280 
methods    of   staining,  142- 

144 

dry  method,  1 66 
Gabbett's,  144 
Gray's,  166-169 
Koch-Ehrlich's,141,166 
NuttalFs     modification, 

144 

Ziehl-Neelsen's,  166 
microscopic    appearance  of, 

280,  281 
organisms  that   simulate  it, 

282,  283 
differential  diagnosis  of,  282- 

284 
staining  of,  in  tissues,  166- 

169 

staining  peculiarities,  281 
Tyndall,  20 
Typhoid  fever,  bacillus  of,  312- 

321 
constant  properties  of,  312, 

313 

cultivation  of,  313-315 
difficulty  in  identifying,  319 
differentiation  from  bacillus 

coli  communis,  821 
experiments  with,  321 
inoculations  with,  316-319 
location  of,  in  tissues,  315, 31 6 
morphology  of,  312 
source  from  which  to  obtain, 

320 

water  as  a  carrier  of,  439,  442, 
443 


VAUGHAN,  429 

T      Vibrio    Metchnikovi,  371- 

375 

characteristics  of,  cultu- 
ral, 372-374 

morphological,  371, 

372 
pathogenesis     of,     374, 

375 
Vibrio    proteus  of  Fintler   and 

Prior,  359-364 
cultivation  of,  360-363 
morphology  of,  359,  360 
pathogenesis  of,  363,  364 
relation  to  cholera   nostras, 

364 
Vibrion  septique,  402-407 


WALDEYEK,  22 
VV    Water,   general     observa- 
tions  upon    bacterio- 
logical study  of,  439 
qualitative  bacteriologi- 
cal analysis  of,  445- 
448 

precautions  in    ob- 
taining    sample, 
445 
preliminary      steps 

in,  445,  446 

quantitative       bacterio- 
logical     analysis 
of,  448-454 
counting     of    colo- 
nies    in,     454- 
458 

apparatus    for, 

455-457 
dilution   of  sample 

in,  452 
obtaining      sample 

for,  449-451 
selection  of  proper 
medium  for,  453, 
454 
source      of      error, 

458 
relation    to    epidemics, 

439,  440 

typhoid   bacilli  in,  442, 
443 


INDEX 


493 


Water,   value  of  bacteriological 

examination  of,  440,  441 
value  of  chemical  examina- 
tion of,  441,  442 
Weigert,  26 
Welch,  247,  257,  260 
Wilde,  23 

Winogradsky,     nitro-monas     of, 
391-394 


Wound  infection.  22-26 
Wurtz's  agar-agar  on  gelatin,  101, 
102 


yOOGLCEA  of  bacteria,  40 
LJ     Zymogenic  bacteria,  30 


YC  88455 


H: 


BIOLOGT 

LIBRARY 

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